One pot desialylation and glycopegylation of therapeutic peptides

ABSTRACT

The present invention provides conjugates between peptides and PEG moieties. The conjugates are linked via an intact glycosyl linking group that is interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from both glycosylated and unglycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto either an amino acid or glycosyl residue on the peptide. Also provided are pharmaceutical formulations including the conjugates. Methods for preparing the conjugates are also within the scope of the invention.

CROSS-REFERENCES TO OTHER APPLICATIONS

The present application is a continuation-in-part of PCT/US06/32649,filed Aug. 21, 2006 and is related to U.S. Provisional PatentApplications 60/746,868, filed May 9, 2006; 60/756,443, filed Jan. 5,2006; 60/733,649, filed Nov. 4, 2005; and 60/730,607, filed Oct. 26,2005, which are incorporated by reference in their entirety for allpurposes

BACKGROUND OF THE INVENTION

The attachment of synthetic polymers to the peptide backbone in anattempt to improve the pharmacokinetic properties of glycoproteintherapeutics is known in the art. An exemplary polymer that has beenconjugated to peptides is poly(ethylene glycol) (“PEG”). The use of PEGto derivatize peptide therapeutics has been demonstrated to reduce theimmunogenicity of the peptides. For example, U.S. Pat. No. 4,179,337(Davis et al.) discloses non-immunogenic polypeptides such as enzymesand peptide hormones coupled to polyethylene glycol (PEG) orpolypropylene glycol. In addition to reduced immunogenicity, theclearance time in circulation is prolonged due to the increased size ofthe PEG-conjugate of the polypeptides in question.

The principal mode of attachment of PEG, and its derivatives, topeptides is a non-specific bonding through a peptide amino acid residue(see e.g., U.S. Pat. No. 4,088,538 U.S. Pat. No. 4,496,689, U.S. Pat.No. 4,414,147, U.S. Pat. No. 4,055,635, and PCT WO 87/00056). Anothermode of attaching PEG to peptides is through the non-specific oxidationof glycosyl residues on a glycopeptide (see e.g., WO 94/05332).

In these non-specific methods, poly(ethylene glycol) is added in arandom, non-specific manner to reactive residues on a peptide backbone.Of course, random addition of PEG molecules has its drawbacks, includinga lack of homogeneity of the final product, and the possibility forreduction in the biological or enzymatic activity of the peptide.Therefore, for the production of therapeutic peptides, a derivitizationstrategy that results in the formation of a specifically labeled,readily characterizable, essentially homogeneous product is superior.Such methods have been developed.

Specifically labeled, homogeneous peptide therapeutics can be producedin vitro through the action of enzymes. Unlike the typical non-specificmethods for attaching a synthetic polymer or other label to a peptide,enzyme-based syntheses have the advantages of regioselectivity andstereoselectivity. Two principal classes of enzymes for use in thesynthesis of labeled peptides are glycosyltransferases (e.g.,sialyltransferases, oligosaccharyltransferases,N-acetylglucosaminyltransferases), and glycosidases. These enzymes canbe used for the specific attachment of sugars which can be subsequentlymodified to comprise a therapeutic moiety. Alternatively,glycosyltransferases and modified glycosidases can be used to directlytransfer modified sugars to a peptide backbone (see e.g., U.S. Pat. No.6,399,336, and U.S. Patent Application Publications 20030040037,20040132640, 20040137557, 20040126838, and 20040142856, each of whichare incorporated by reference herein). Methods combining both chemicaland enzymatic synthetic elements are also known (see e.g., Yamamoto etal. Carbohydr. Res. 305: 415-422 (1998) and U.S. Patent ApplicationPublication 20040137557 which is incorporated herein by reference).

In response to the need for improved therapeutic peptide, the presentinvention provides a glycopegylated peptide that is therapeuticallyactive and which has pharmacokinetic parameters and properties that areimproved relative to an identical, or closely analogous, peptide that isnot glycopegylated. Furthermore, the invention provides method forproducing cost effectively and on an industrial scale the improvedpeptides of the invention.

SUMMARY OF THE INVENTION

It has now been discovered that the controlled modification of a peptidewith one or more sugar moiety, either unmodified or modified, e.g., withpoly(ethylene glycol) moieties, affords a novel peptide conjugate withpharmacokinetic properties that are improved relative to thecorresponding native unmodified, e.g., (un-pegylated), peptide.Furthermore, cost effective methods for reliable and reproducibleproduction of the peptide conjugates of the invention have beendiscovered and developed.

Quite surprisingly, it has been discovered that a reaction mixture thatincludes both a degradative enzyme (e.g., sialidase) and a syntheticenzyme (e.g., sialyltransferase) can be used to transfer a sugar residue(e.g., PEGylated sialic acid) onto a peptide. Contrary to common wisdom,the degradative enzyme does not significantly interfere with transfer ofthe sugar residue to the peptide. Thus, the instant discovery providesfor added convenience in forming peptide conjugates, because thedegradative and synthetic enzymes can be utilized simultaneously withoutthe need to remove the degradative enzyme prior to performing thesynthesis step.

In a first aspect, the invention provides a method alteringglycosylation of a peptide. The method includes, (a) contacting thepeptide with a sialidase, thereby removing at least one sialic acidmoiety from the peptide. The peptide can be partially or whollydesialylated as discussed above. In step (b), the product from step (a)is contacted with at least one glycosyltransferase and at least onesugar donor. The at least one sugar donor is a substrate for the atleast one glycosyltransferase. Preferably, step (b) results in transferof a sugar residue from the donor onto an amino acid or glycosyl residueof the peptide. The sugar residue can be a modified or unmodified sugarresidue. The conditions of step (b) are maintained until a desired levelof glycosylation of the peptide is achieved, as discussed above.Following step (b), the sialidase content of the reaction mixture isreduced and preferably, essentially all of the sialidase is removed. Inan exemplary embodiment, the removal of sialidase is effected by achromatography (e.g., ion exchange, e.g., anion exchange) or filtrationstep (e.g., nanofiltration). When the sialidase is removed, theresulting glycosylated peptide (i.e., produced in step (a)) isoptionally contacted with a further at least one sugar donor and atleast one glycosyltransferase, transferring at least one sugar residueonto an amino acid or glycosyl moiety of the peptide. In a preferredembodiment, the sugar residue is sialic acid. In a still furtherpreferred embodiment, the sugar donor is CMP-sialic acid. Theglycosyltransferase is typically a sialyltransferase.

An exemplary modified sugar donor includes a sugar residue modified witha polymer (e.g., a water-soluble polymer, e.g., PEG), a therapeuticagent, a diagnostic agent, a bioactive agent and/or a peptide.

The process set forth above is of use for preparing “glycopegylated”peptides in which a glycosyltransferase (or enzyme havingglycosyltransferase activity) transfers to an amino acid or glycosylresidue of the peptide a sugar moiety modified with a PEG moiety. As setforth herein, the process allows for control of the degree ofglycopegylation of the peptide by control of the speed of theglycopegylation reaction and the time the reaction is allowed to run.When the reaction is completed, exposed Gal residues on theglycopegylated peptide can be “capped” using sialic acid.

In an exemplary embodiment, “glycopegylated” peptide molecules of theinvention are produced by the enzyme mediated formation of a conjugatebetween a glycosylated or non-glycosylated peptide and an enzymaticallytransferable saccharyl moiety that includes a modifying group, such as apolymeric modifying group such as poly(ethylene glycol), within itsstructure. The PEG moiety is attached to the saccharyl moiety directly(i.e., through a single group formed by the reaction of two reactivegroups) or through a linker moiety, e.g., substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, etc.

Thus, in one aspect, the present invention provides a conjugate betweena PEG moiety, e.g., PEG and a peptide that has an in vivo activitysimilar or otherwise analogous to art-recognized peptide. In theconjugate of the invention, the PEG moiety is covalently attached to thepeptide via an intact glycosyl linking group. Exemplary intact glycosyllinking groups include sialic acid moieties that are derivatized withPEG.

The polymeric modifying group can be attached at any position of aglycosyl moiety of a peptide. Moreover, the polymeric modifying groupcan be bound to a glycosyl residue at any position in the amino acidsequence of a wild type or mutant peptide.

In an exemplary embodiment, the invention provides a peptide that isconjugated through a glycosyl linking group to a polymeric modifyinggroup. Exemplary peptide conjugates include a glycosyl linking grouphaving a formula selected from:

In Formulae I and II, R² is H, CH₂OR⁷, COOR⁷, COO⁻ or OR⁷, in which R⁷represents H, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl. The symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR^(B),NHC(O)R⁹. The index d is 0 or 1. R⁸ and R⁹ are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl or sialic acid. At least one of R³, R⁴, R⁵, R⁶ or R^(6′)includes the polymeric modifying group e.g., PEG. In an exemplaryembodiment, R⁶ and R^(6′), together with the carbon to which they areattached are components of the side chain of a sialyl moiety. In afurther exemplary embodiment, this side chain is functionalized with thepolymeric modifying group.

In an exemplary embodiment, the polymeric modifying group is bound tothe glycosyl linking group, generally through a heteroatom on theglycosyl core (e.g., N, O), through a linker, L, as shown below:

R¹ is the polymeric modifying group and L is selected from a bond and alinking group. The index w represents an integer selected from 1-6,preferably 1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety. Another exemplary linking group is an aminoacid residue (e.g., cysteine, serine, lysine, and short oligopeptides,e.g., Lys-Lys, Lys-Lys-Lys, Cys-Lys, Ser-Lys, etc.)

When L is a bond, it is formed by reaction of a reactive functionalgroup on a precursor of R¹ and a reactive functional group ofcomplementary reactivity on a precursor of the glycosyl linking group.When L is a non-zero order linking group, L can be in place on theglycosyl moiety prior to reaction with the R¹ precursor. Alternatively,the precursors of R¹ and L can be incorporated into a preformed cassettethat is subsequently attached to the glycosyl moiety. As set forthherein, the selection and preparation of precursors with appropriatereactive functional groups is within the ability of those skilled in theart. Moreover, coupling of the precursors proceeds by chemistry that iswell understood in the art.

In an exemplary embodiment L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying moiety is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. Amino acid analogs, as defined herein, arealso of use as linker components. The amino acid may be modified with anadditional component of a linker, e.g., alkyl, heteroalkyl, covalentlyattached through an acyl linkage, for example, an amide or urethaneformed through an amine moiety of the amino acid residue.

In an exemplary embodiment, the glycosyl linking group has a structureaccording to Formula I and R⁵ includes the polymeric modifying group. Inanother exemplary embodiment, R⁵ includes both the polymeric modifyinggroup and a linker, L, joining the polymeric modifying group to theglycosyl core. L can be a linear or branched structure. Similarly, thepolymeric modifying group can be branched or linear.

The polymeric modifying group comprises two or more repeating units thatcan be water-soluble or essentially insoluble in water. Exemplarywater-soluble polymers of use in the compounds of the invention includePEG, e.g., m-PEG, PPG, e.g., m-PPG, polysialic acid, polyglutamate,polyaspartate, polylysine, polyethyeleneimine, biodegradable polymers(e.g., polylactide, polyglyceride), and functionalized PEG, e.g.,terminal-functionalized PEG.

The glycosyl core of the glycosyl linking groups of use in the peptideconjugates are selected from both natural and unnatural furanoses andpyranoses. The unnatural saccharides optionally include an alkylated oracylated hydroxyl and/or amine moiety, e.g., ethers, esters and amidesubstituents on the ring. Other unnatural saccharides include an H,hydroxyl, ether, ester or amide substituent at a position on the ring atwhich such a substituent is not present in the natural saccharide.Alternatively, the carbohydrate is missing a substituent that would befound in the carbohydrate from which its name is derived, e.g., deoxysugars. Still further exemplary unnatural sugars include both oxidized(e.g., -onic and -uronic acids) and reduced (sugar alcohols)carbohydrates. The sugar moiety can be a mono-, oligo- orpoly-saccharide.

Exemplary natural sugars of use as components of glycosyl linking groupsin the present invention include glucose, glucosamine, galactose,galactosamine, fucose, mannose, mannosamine, xylanose, ribose, N-acetylglucose, N-acetyl glucosamine, N-acetyl galactose, N-acetylgalactosamine, and sialic acid.

In one embodiment, the present invention provides a peptide conjugatecomprising the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched poly(ethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In another aspect, the invention provides a peptide conjugate comprisinga peptide. The conjugate also comprises a glycosyl linking group,wherein the glycosyl linking group is attached to an amino acid residueof said peptide, and wherein said glycosyl linking group comprises asialyl linking group having a formula which is a member selected from:

are modifying groups. R² is a member selected from H, CH₂OR⁷, COOR⁷,COO⁻ and OR⁷. R⁷ is a member selected from H, substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. R³ andR⁴ are members independently selected from H, substituted orunsubstituted alkyl, OR⁸, and NHC(O)R⁹. R⁸ and R⁹ are independentlyselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl and sialyl. L^(a) is a linker selected from abond, substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl. X⁵, R¹⁶ and R¹⁷ are independently selectedfrom non-reactive group and polymeric arms (e.g. PEG). X² and X⁴ areindependently selected linkage fragments joining polymeric moieties R¹⁶and R¹⁷ to C. The index j is an integer selected from 1 to 15.

In another exemplary embodiment, the polymeric modifying group has astructure according to the following formula:

in which the indices m and n are integers independently selected from 0to 5000. A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, A¹⁰ and A¹¹ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,—NA¹²A¹³, —OA¹² and —SiA¹²A¹³. A¹² and A¹³ are members independentlyselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

In an exemplary embodiment, the polymeric modifying group has astructure according to the following formulae:

In another exemplary embodiment according to the formula above, thepolymeric modifying group has a structure according to the followingformula:

In an exemplary embodiment, A¹ and A² are each members selected from —OHand —OCH₃.

Exemplary polymeric modifying groups according to this embodimentinclude:

The invention provides a peptide conjugate comprising a peptide. Theconjugate also comprises a glycosyl linking group, wherein the glycosyllinking group is attached to an amino acid residue of the peptide, andwherein the glycosyl linking group comprises a sialyl linking grouphaving the formula:

is a modifying group. The index s is an integer selected from 1 to 20.The index f is an integer selected from 1 to 2500. Q is a memberselected from H and substituted or unsubstituted C₁-C₆ alkyl.

In an exemplary embodiment, the invention provides a modified sugarhaving the following formula:

The present invention provides methods of forming conjugates ofpeptides. The methods include contacting a peptide with a modified sugardonor that bears a modifying group covalently attached to a sugar. Themodified sugar moiety is transferred from the donor onto an amino acidor glycosyl residue of the peptide by the action of an enzyme.Representative enzymes include, but are not limited to,glycosyltransferases, e.g., sialyltransferases. The method includescontacting the peptide with: a) a modified sugar donor; and b) an enzymecapable of transferring a modified sugar moiety from the modified sugardonor onto an amino acid or glycosyl residue of the peptide, underconditions appropriate to transfer a modified sugar moiety from thedonor to an amino acid or glycosyl residue of the peptide, therebysynthesizing said peptide conjugate.

In a preferred embodiment, prior to step a), the peptide is contactedwith a sialidase, thereby removing at least a portion of the sialic acidon the peptide.

In another preferred embodiment, the peptide is contacted with asialidase, a glycosyltransferase and a modified sugar donor. In thisembodiment, the peptide is in contact with the sialidase,glycosyltransferase and modified sugar donor essentially simultaneously,no matter the order of addition of the various components. The reactionis carried out under conditions appropriate for the sialidase to removea sialic acid residue from the peptide; and the glycosyltransferase totransfer a modified sugar moiety from the modified sugar donor to anamino acid or glycosyl residue of the peptide.

In another preferred embodiment, the desialylation and conjugation areperformed in the same vessel, and the desialylated peptide is preferablynot purified prior to the conjugation step. In another exemplaryembodiment, the method further comprises a ‘capping’ step involvingsialylation of the peptide conjugate. This step is performed in the samereaction vessel that contains the sialidase, sialyltransferase andmodified sugar donor without prior purification.

In another preferred embodiment, the desialylation of the peptide isperformed, and the asialo peptide is purified. The purified asialopeptide is then subjected to conjugation reaction conditions. In anotherexemplary embodiment, the method further comprises a ‘capping’ stepinvolving sialylation of the peptide conjugate. This step is performedin the same reaction vessel that contains the sialidase,sialyltransferase and modified sugar donor without prior purification.

In another exemplary embodiment, the capping step, sialylation of thepeptide conjugate, is performed in the same reaction vessel thatcontains the sialidase, sialyltransferase and modified sugar donorwithout prior purification.

In an exemplary embodiment, the contacting is for a time less than 20hours, preferably less than 16 hours, more preferably less than 12hours, even more preferably less than 8 hours, and still more preferablyless than 4 hours.

In a further aspect, the present invention provides a peptide conjugatereaction mixture. The reaction mixture comprises: a) a sialidase; b) anenzyme which is a member selected from glycosyltransferase,exoglycosidase and endoglycosidase; c) a modified sugar; and d) apeptide.

In another exemplary embodiment, the ratio of the sialidase to thepeptide is selected from 0.1 U/L:2 mg/mL to 10 U/L:1 mg/mL, preferably0.5 U/L:2 mg/mL, more preferably 1.0 U/L:2 mg/mL, even more preferably10 U/L:2 mg/mL, still more preferably 0.1 U/L:1 mg/mL, more preferably0.5 U/L:1 mg/mL, even more preferably 1.0 U/L:1 mg/mL, and still morepreferably 10 U/L:1 mg/mL.

In an exemplary embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%or 80% of said peptide conjugate includes at most two PEG moieties. ThePEG moieties can be added in a one-pot process, or they can be addedafter the asialo peptide is purified.

In another exemplary embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%,70% or 80% of the peptide conjugate include at most one PEG moiety. ThePEG moiety can be added in a one-pot process, or it can be added afterthe asialo peptide is purified.

In a further exemplary embodiment, the method further comprises“capping”, or adding sialic acid to the peptide conjugate. In anotherexemplary embodiment, sialidase is added, followed by a delay of 30 min,1 hour, 1.5 hours, or 2 hours, followed by the addition of theglycosyltransferase, exoglycosidase, or endoglycosidase.

In another exemplary embodiment, sialidase is added, followed by a delayof 30 min, 1 hour, 1.5 hours, or 2 hours, followed by the addition ofthe glycosyltransfase, exoglycosidase, or endoglycosidase. Other objectsand advantages of the invention will be apparent to those of skill inthe art from the detailed description that follows.

In another exemplary embodiment, the method includes: (a) contacting apeptide comprising a glycosyl group selected from:

with a modified sugar having the formula:

and an appropriate transferase which transfers the glycosyl linkinggroup onto a member selected from the GalNAc, Gal and the Sia of saidglycosyl group, under conditions appropriate for said transfer. Anexemplary modified sugar is CMP-sialic acid modified, through a linkermoiety, with a polymer, e.g., a straight chain or branched poly(ethyleneglycol) moiety.

The peptide can be acquired from essentially any source, however, in oneembodiment, prior to being modified as discussed above, the peptide isexpressed in a suitable host. Mammalian (e.g., BHK, CHO), bacteria(e.g., E. coli) and insect cells (e.g., Sf-9) are exemplary expressionsystems providing peptides of use in the compositions and methods setforth herein.

In exemplary embodiments, a peptide conjugate may be administered topatients for the treatment of a tissue injury such as ischemia, trauma,inflammation, or contact with toxic substances. In other exemplaryembodiments, a peptide conjugate may be administered to patients for thetreatment of a patient having Hemophilia A, a patient with Hemophilia B,a patient having Hemophilia A, wherein the patient also has antibodiesto Factor VIII, a patient having Hemophilia B, wherein the patient alsohas antibodies to Factor IX, and a patient having liver cirrhosis.

In another exemplary embodiment, a peptide conjugate may be administeredto patients for the treatment of bleeding in emergencies, electivesurgery, cardiac surgery, spinal surgery, liver transplantation, partialhepatectomies, pelvic-acetabular fracture reconstruction, and allogeneicstem cell transplantation. In another exemplary embodiment, a peptideconjugate may be administered to patients for the treatment of acuteintracerebral haemorrhage, traumatic brain injury, variceal bleedingsand upper gastrointestinal bleeding.

In another aspect, the invention provides a pharmaceutical formulationcomprising a peptide conjugate and a pharmaceutically acceptablecarrier.

In the peptide conjugate, essentially each of the amino acid residues towhich the glycosyl linking group or modifying group is bound has thesame structure. For example, if one peptide includes a Thr linkedglycosyl residue, at least about 70%, 80%, 90%, 95%, 97%, 99%, 99.2%,99.4%, 99.6%, or more preferably 99.8% of the peptides in the populationwill have the same glycosyl linking group covalently bound to the sameThr residue.

Other objects and advantages of the invention will be apparent to thoseof skill in the art from the detailed description that follows.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates exemplary modified sialic acid nucleotides useful inthe practice of the invention. A. Structure of exemplary branched (e.g.,30 KDa, 40 KDa) CMP-sialic acid-PEG sugar nucleotides. B. Structure oflinear Factor VIIa-SA-PEG-10 KDa.

FIG. 2 is a synthetic scheme for producing an exemplary PEG-glycosyllinking group precursor (modified sugar) of use in preparing theconjugates of the invention.

FIG. 3 is a table providing exemplary sialyltransferases of use informing the glycoconjugates of the invention, e.g., to glycoPEGylatepeptides with a modified sialic acid.

FIG. 4, comprising FIGS. 4A to 4E, sets forth exemplary schemes forremodeling glycan structures on Factor VII and Factor VIIa. FIG. 4A is adiagram depicting the Factor VII and Factor VIIa peptides indicating theresidues which bind to glycans contemplated for remodeling. FIG. 4B is adiagram depicting the Factor VII and Factor VIIa peptides A (solid line)and B (dotted line) indicating the residues which bind to glycanscontemplated for remodeling, and the formulas for the glycans. FIGS. 4Cto 4E are diagrams of contemplated remodeling steps of the glycan of thepeptide in FIG. 4B based on the type of cell the peptide is expressed inand the desired remodeled glycan structure.

FIG. 5, comprising FIGS. 5A and 5B, is an exemplary nucleotide andcorresponding amino acid sequence of Factor VIIa (SEQ ID NOS: 1 and 2,respectively).

FIG. 6 is an image of an isoelectric focusing gel (pH 3-7) ofasialo-Factor VIIa. Lane 1 is Factor VIIa; lanes 2-5 are asialo-FactorVIIa.

FIG. 7 is a graph of a MALDI spectra of Factor VIIa.

FIG. 8 is a graph of a MALDI spectra of Factor VIIa-SA-PEG-1 KDa.

FIG. 9 is a graph depicting a MALDI spectra of Factor VIIa-SA-PEG-10KDa.

FIG. 10 is an image of an SDS-PAGE gel of PEGylated Factor VIIa. Lane 1is asialo-Factor VIIa. Lane 2 is the product of the reaction ofasialo-Factor VIIa and CMP-SA-PEG-1 KDa with ST3Gal3 after 48 hr. Lane 3is the product of the reaction of asialo-Factor VIIa and CMP-SA-PEG-1KDa with ST3Gal3 after 48 hr. Lane 4 is the product of the reaction ofasialo-Factor VIIa and CMP-SA-PEG-10 KDa with ST3Gal3 at 96 hr.

FIG. 11 A-B shows simultaneous desialylation, with less sialidase, andPEGylation. These figures highlight that capping in the presence ofsialidase is efficient. FIG. 11A shows the reaction course when thesialidase is at a level of 0.5 U/L. Lane 1 corresponds to native FactorVIIa while Lane 2 is asialo Factor VIIa. From Lane 3 to Lane 7, there isan increasing amount of PEGylated product as time progresses. In Lane 3,the major product is monoPEGylated (see spot at 64), while aliquotsassayed at later times show the formation and increasing amounts of di(see spot just below 97), tri (see spot just above 97), and higherPEGylated products. Lanes 8 and 9 show the results of ‘capping’, oradding sialic acid, to the reaction. When the reaction is capped, theextent of reaction is stopped, as can be seen from the similar PEGylatedproduct distribution found in Lanes 5, 8 and 9. FIG. 11B shows thereaction course when the sialidase is at a level of 0.1 U/L.

FIG. 12 shows the situation when the sialidase and theglycosyltransferase are added at the same time.

FIG. 13 is a table of the peptides to which one or more glycosyl linkinggroups can be attached to order to provide the peptide conjugates of theinvention.

FIGS. 14 A and B displays chromatograms showing the results of HPLCexperiments. FIG. 14A displays labeled chromatograms of FactorVIIa-SA-PEG-10 KDa (top) and native Factor VIIa control (bottom)analyzed by the light chain method. The separation of LC (light chain),1×10 KDa-PEG-LC, 2×10 KDa-PEG-LC, and 3×10 KDa-PEG-LC from otherproducts is shown. FIG. 14B displays labeled chromatograms of FactorVIIa-SA-PEG-10 KDa (top) and native Factor VIIa control (bottom)analyzed by heavy chain method. The separation of HC (heavy chain), 1×10KDa-PEG-HC, 2×10 KDa-PEG-HC, and 3×10 KDa-PEG-HC from other products isshown.

FIGS. 15 A and B displays chromatograms showing the results of HPLCexperiments. FIG. 15A displays labeled chromatograms of reduced nativeFactor VIIa control (top) and reduced Factor VIIa-SA-PEG-40 KDa (bottom)analyzed by the light chain method. The separation of LC (light chain),1×40 KDa-PEG-LC, 2×40 KDa-PEG-LC, and 3×40 KDa-PEG-LC from otherproducts is shown. FIG. 15B displays labeled chromatograms of reducednative Factor VIIa control (top) and Factor VIIa-SA-PEG-40 KDa (bottom)analyzed by the heavy chain method. The separation of HC (heavy chain),1×40 KDa-PEG-HC, 2×40 KDa-PEG-HC, and 3×40 KDa-PEG-HC from otherproducts is shown.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTSAbbreviations

PEG, poly(ethyleneglycol); PPG, poly(propyleneglycol); Ara, arabinosyl;Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc,N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl;Man, mannosyl; ManAc, mannosaminyl acetate; Xyl, xylosyl; NeuAc, sialylor N-acetylneuraminyl; Sia, sialyl or N-acetylneuraminyl; andderivatives and analogues thereof.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

All oligosaccharides described herein are described with the name orabbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond (1 or2), the ring position of the reducing saccharide involved in the bond(2, 3, 4, 6 or 8), and then the name or abbreviation of the reducingsaccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. Fora review of standard glycobiology nomenclature, see, Essentials ofGlycobiology Varki et al. eds. CSHL Press (1999).

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

The term “sialic acid” or “sialyl” refers to any member of a family ofnine-carbon carboxylated sugars. The most common member of the sialicacid family is N-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also included. Amino acidsthat are not gene-encoded may also be used in the present invention.Furthermore, amino acids that have been modified to include reactivegroups, glycosylation sites, polymers, therapeutic moieties,biomolecules and the like may also be used in the invention. All of theamino acids used in the present invention may be either the D- orL-isomer. The L-isomer is generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. As usedherein, “peptide” refers to both glycosylated and unglycosylatedpeptides. Also included are peptides that are incompletely glycosylatedby a system that expresses the peptide. For a general review, see,Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDESAND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983). A listing of some of the peptides of the invention is providedin FIG. 13.

The term “peptide conjugate,” refers to species of the invention inwhich a peptide is conjugated with a modified sugar as set forth herein.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

As used herein, the term “modified sugar,” or “modified sugar residue”,refers to a naturally- or non-naturally-occurring carbohydrate that isenzymatically added onto an amino acid or a glycosyl residue of apeptide in a process of the invention. The modified sugar is selectedfrom enzyme substrates including, but not limited to sugar nucleotides(mono-, di-, and tri-phosphates), activated sugars (e.g., glycosylhalides, glycosyl mesylates) and sugars that are neither activated nornucleotides. The “modified sugar” is covalently functionalized with a“modifying group.” Useful modifying groups include, but are not limitedto, PEG moieties, therapeutic moieties, diagnostic moieties,biomolecules and the like. The modifying group is preferably not anaturally occurring, or an unmodified carbohydrate. The locus offunctionalization with the modifying group is selected such that it doesnot prevent the “modified sugar” from being added enzymatically to apeptide.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesof be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol). Poly(ethylene imine) is an exemplary polyamine,and poly(acrylic) acid is a representative poly(carboxylic acid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(m) in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, within about 2 toabout 300 loci for attachment, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers of ethylene glycol and propylene glycol and the like,poly(oxyethylated polyol), poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide),poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene,polyoxazoline, poly(N-acryloylmorpholine), such as described in U.S.Pat. No. 5,629,384, which is incorporated by reference herein in itsentirety, and copolymers, terpolymers, and mixtures thereof. Althoughthe molecular weight of each chain of the polymer backbone can vary, itis typically in the range of from about 100 Da to about 100,000 Da,often from about 6,000 Da to about 80,000 Da.

The “area under the curve” or “AUC”, as used herein in the context ofadministering a peptide drug to a patient, is defined as total areaunder the curve that describes the concentration of drug in systemiccirculation in the patient as a function of time from zero to infinity.

The term “half-life” or “t½”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the timerequired for plasma concentration of a drug in a patient to be reducedby one half. There may be more than one half-life associated with thepeptide drug depending on multiple clearance mechanisms, redistribution,and other mechanisms well known in the art. Usually, alpha and betahalf-lives are defined such that the alpha phase is associated withredistribution, and the beta phase is associated with clearance.However, with protein drugs that are, for the most part, confined to thebloodstream, there can be at least two clearance half-lives. For someglycosylated peptides, rapid beta phase clearance may be mediated viareceptors on macrophages, or endothelial cells that recognize terminalgalactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, orfucose. Slower beta phase clearance may occur via renal glomerularfiltration for molecules with an effective radius <2 nm (approximately68 kD) and/or specific or non-specific uptake and metabolism in tissues.GlycoPEGylation may cap terminal sugars (e.g., galactose orN-acetylgalactosamine) and thereby block rapid alpha phase clearance viareceptors that recognize these sugars. It may also confer a largereffective radius and thereby decrease the volume of distribution andtissue uptake, thereby prolonging the late beta phase. Thus, the preciseimpact of glycoPEGylation on alpha phase and beta phase half-lives mayvary depending upon the size, state of glycosylation, and otherparameters, as is well known in the art. Further explanation of“half-life” is found in Pharmaceutical Biotechnology (1997, DFACrommelin and R D Sindelar, eds., Harwood Publishers, Amsterdam, pp101-120).

The term “glycoconjugation,” as used herein, refers to the enzymaticallymediated conjugation of a modified sugar species to an amino acid orglycosyl residue of a polypeptide, e.g., a G-CSF peptide of the presentinvention. A subgenus of “glycoconjugation” is “glyco-PEGylation,” inwhich the modifying group of the modified sugar is poly(ethyleneglycol), and alkyl derivative (e.g., m-PEG) or reactive derivative(e.g., H₂N-PEG, HOOC-PEG) thereof.

The terms “large-scale” and “industrial-scale” are used interchangeablyand refer to a reaction cycle that produces at least about 250 mg,preferably at least about 500 mg, and more preferably at least about 1gram of glycoconjugate at the completion of a single reaction cycle.

The term, “glycosyl linking group,” as used herein refers to a glycosylresidue to which a modifying group (e.g., PEG moiety, therapeuticmoiety, biomolecule) is covalently attached; the glycosyl linking groupjoins the modifying group to the remainder of the conjugate. In themethods of the invention, the “glycosyl linking group” becomescovalently attached to a glycosylated or unglycosylated peptide, therebylinking the agent to an amino acid and/or glycosyl residue on thepeptide. A “glycosyl linking group” is generally derived from a“modified sugar” by the enzymatic attachment of the “modified sugar” toan amino acid and/or glycosyl residue of the peptide. The glycosyllinking group can be a saccharide-derived structure that is degradedduring formation of modifying group-modified sugar cassette (e.g.,oxidation→Schiff base farmation→reduction), or the glycosyl linkinggroup may be intact. An “intact glycosyl linking group” refers to alinking group that is derived from a glycosyl moiety in which thesaccharide monomer that links the modifying group and to the remainderof the conjugate is not degraded, e.g., oxidized, e.g., by sodiummetaperiodate. “Intact glycosyl linking groups” of the invention may bederived from a naturally occurring oligosaccharide by addition ofglycosyl unit(s) or removal of one or more glycosyl unit from a parentsaccharide structure.

The term, “non-glycosidic modifying group”, as used herein, refers tomodifying groups which do not include a naturally occurring sugar linkeddirectly to the glycosyl linking group.

The term “targeting moiety,” as used herein, refers to species that willselectively localize in a particular tissue or region of the body. Thelocalization is mediated by specific recognition of moleculardeterminants, molecular size of the targeting agent or conjugate, ionicinteractions, hydrophobic interactions and the like. Other mechanisms oftargeting an agent to a particular tissue or region are known to thoseof skill in the art. Exemplary targeting moieties include antibodies,antibody fragments, transferrin, HS-glycoprotein, coagulation factors,serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the like.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is bound to a carrier, e.g, multivalentagents. Therapeutic moiety also includes proteins and constructs thatinclude proteins. Exemplary proteins include, but are not limited to,Granulocyte Colony Stimulating Factor (GCSF), Granulocyte MacrophageColony Stimulating Factor (GMCSF), Interferon (e.g., Interferon-α, -β,-γ), Interleukin (e.g., Interleukin II), serum proteins (e.g., FactorsVII, VIIa, VIII, IX, and X), Human Chorionic Gonadotropin (HCG),Follicle Stimulating Hormone (FSH) and Lutenizing Hormone (LH) andantibody fusion proteins (e.g. Tumor Necrosis Factor Receptor ((TNFR)/Fcdomain fusion protein)).

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the conjugates'activity and is non-reactive with the subject's immune systems. Examplesinclude, but are not limited to, any of the standard pharmaceuticalcarriers such as a phosphate buffered saline solution, water, emulsionssuch as oil/water emulsion, and various types of wetting agents. Othercarriers may also include sterile solutions, tablets including coatedtablets and capsules. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well known conventionalmethods.

As used herein, “administering,” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, or the implantation of a slow-releasedevice e.g., a mini-osmotic pump, to the subject. Administration is byany route including parenteral, and transmucosal (e.g., oral, nasal,vaginal, rectal, or transdermal). Parenteral administration includes,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial.Moreover, where injection is to treat a tumor, e.g., induce apoptosis,administration may be directly to the tumor and/or into tissuessurrounding the tumor. Other modes of delivery include, but are notlimited to, the use of liposomal formulations, intravenous infusion,transdermal patches, etc.

The term “ameliorating” or “ameliorate” refers to any indicia of successin the treatment of a pathology or condition, including any objective orsubjective parameter such as abatement, remission or diminishing ofsymptoms or an improvement in a patient's physical or mental well-being.Amelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination and/or apsychiatric evaluation.

The term “therapy” refers to “treating” or “treatment” of a disease orcondition including preventing the disease or condition from occurringin an animal that may be predisposed to the disease but does not yetexperience or exhibit symptoms of the disease (prophylactic treatment),inhibiting the disease (slowing or arresting its development), providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment), and relieving the disease (causing regression ofthe disease).

The term “effective amount” or “an amount effective to” or a“therapeutically effective amount” or any grammatically equivalent termmeans the amount that, when administered to an animal for treating adisease, is sufficient to effect treatment for that disease.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents which normally accompany the material in the mixture used toprepare the peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

When the peptide conjugates are more than about 90% pure, their puritiesare also preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of peptide conjugates of the invention inwhich a selected percentage of the modified sugars added to a peptideare added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the modified sugars are conjugated. Thus,in a peptide conjugate of the invention in which each modified sugarmoiety is conjugated to an acceptor site having the same structure asthe acceptor site to which every other modified sugar is conjugated, thepeptide conjugate is said to be about 100% homogeneous. Homogeneity istypically expressed as a range. The lower end of the range ofhomogeneity for the peptide conjugates is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (LC-MS), matrix assisted laserdesorption mass time of flight spectrometry (MALDITOF), capillaryelectrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all (as defined below) ofthe Galβ1,4-GlcNAc-R and sialylated analogues thereof are fucosylated ina peptide conjugate of the invention. In the fucosylated structures setforth herein, the Fuc-GlcNAc linkage is generally α1,6 or α1,3, withα1,6 generally preferred. It will be understood by one of skill in theart, that the starting material may contain glycosylated acceptormoieties (e.g., fucosylated Galβ1,4-GlcNAc-R moieties). Thus, thecalculated percent glycosylation will include acceptor moieties that areglycosylated by the methods of the invention, as well as those acceptormoieties already glycosylated in the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularglycosyltransferase are glycosylated.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R″″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R″', —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R′″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R¹ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R′″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(u)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and u is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(z)—X—(CR″R′″)_(d)—, where z and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The invention is meant to include salts of the compounds of theinvention which are prepared with relatively nontoxic acids or bases,depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of base addition salts include sodium, potassium, lithium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acidaddition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science 66: 1-19 (1977)). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompounds in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

“Salt counterion”, as used herein, refers to positively charged ionsthat associate with a compound of the invention when one of its moietiesis negatively charged (e.g. COO—). Examples of salt counterions includeH⁺, H₃O⁺, ammonium, potassium, calcium, lithium, magnesium and sodium.

As used herein, the term “CMP-SA-PEG” is a cytidine monophosphatemolecule which is conjugated to a sialic acid which comprises apolyethylene glycol moiety. If a length of the polyethylene glycol chainis not specified, then any PEG chain length is possible (e.g. 1 KDa, 2KDa, 5 KDa, 10 KDa, 20 KDa, 30 KDa, 40 KDa). An exemplary CMP-SA-PEG iscompound 5 in Scheme 1.

I. Introduction

To improve the effectiveness of a recombinant peptide used fortherapeutic purposes, the present invention provides conjugates ofglycosylated and unglycosylated peptides with a modifying group. Themodifying groups can be selected from polymeric modifying groups suchas, e.g., PEG (m-PEG), PPG (m-PPG), etc., therapeutic moieties,diagnostic moieties, targeting moieties and the like. Modification ofthe peptides, e.g., with a water-soluble polymeric modifying group canimprove the stability and retention time of the recombinant peptide in apatient's circulation, and/or reduce the antigenicity of recombinantpeptide.

The peptide conjugates of the invention can be formed by the enzymaticattachment of a modified sugar to the glycosylated or unglycosylatedpeptide. A glycosylation site and/or a modified glycosyl group providesa locus for conjugating a modified sugar bearing a modifying group tothe peptide, e.g., by glycoconjugation.

The methods of the invention also make it possible to assemble peptideconjugates and glycopeptide conjugates that have a substantiallyhomogeneous derivatization pattern. The enzymes used in the inventionare generally selective for a particular amino acid residue, combinationof amino acid residues, particular glycosyl residues, or combination ofglycosyl residues of the peptide. The methods are also practical forlarge-scale production of peptide conjugates. Thus, the methods of theinvention provide a practical means for large-scale preparation ofpeptide conjugates having preselected uniform derivatization patterns.The methods are particularly well suited for modification of therapeuticpeptides, including but not limited to, glycopeptides that areincompletely glycosylated during production in cell culture cells (e.g.,mammalian cells, insect cells, plant cells, fungal cells, yeast cells,or prokaryotic cells) or transgenic plants or animals.

The peptide conjugates can be produced as pharmaceutical formulationscomprising a peptide conjugate as well as a pharmaceutically acceptablecarrier. The peptide conjugates may be administered to a patientselected from the group consisting of a hemophiliac patient having ableeding episode, a patient having Hemophilia A, a patient withHemophilia B, a patient having Hemophilia A, wherein the patient alsohas antibodies to Factor VIII, a patient having Hemophilia B, whereinthe patient also has antibodies to Factor IX, a patient having livercirrhosis, a cirrhotic patient having an orthotopic liver transplant, acirrhotic patient having upper gastrointestinal bleeding, a patienthaving a bone marrow transplant, a patient having a liver resection, apatient having a partial hepatectomy, a patient undergoingpelvic-acetabular fracture reconstruction, a patient bleeding from anacute intercerebral hemmorage, a patient undergoing allogeneic stem celltransplantation, a patient bleeding from traumatic brain injury, apatient bleeding in an emergency, a patient having bleeding from trauma,a patient undergoing variceal bleeding, a patient bleeding from electivesurgery, a patient bleeding from cardiac surgery, a patient bleedingfrom spinal surgery, a liver resection a liver resection a liverresection. In an exemplary embodiment, the patient is a human patient.

The present invention also provides conjugates of glycosylated andunglycosylated peptides with increased therapeutic half-life due to, forexample, reduced clearance rate, or reduced rate of uptake by the immuneor reticuloendothelial system (RES). Moreover, the methods of theinvention provide a means for masking antigenic determinants onpeptides, thus reducing or eliminating a host immune response againstthe peptide. Selective attachment of targeting agents can also be usedto target a peptide to a particular tissue or cell surface receptor thatis specific for the particular targeting agent.

Determining optimal conditions for the preparation of peptide conjugateswith water-soluble polymers, e.g., involves the optimization of numerousparameters, which are dependent on the identity of the peptide and ofthe water-soluble polymer. For example, when the polymer ispoly(ethylene glycol), e.g., a branched poly(ethylene glycol), a balanceis preferably established between the amount of polymer utilized in thereaction and the viscosity of the reaction mixture attributable to thepresence of the polymer: if the polymer is too highly concentrated, thereaction mixture becomes viscous, slowing the rate of mass transfer andreaction.

Furthermore, though it is intuitively apparent to add an excess ofenzyme, the present inventors have recognized that, when the enzyme ispresent in too great of an excess, the excess enzyme becomes acontaminant whose removal requires extra purification steps and materialand unnecessarily increases the cost of the final product.

Moreover, it is generally desired to produce a peptide with a controlledlevel of modification. In some instances, it is desirable to add onemodified sugar preferentially. In other instances, it is desirable toadd two modified sugars preferentially. Thus, the reaction conditionsare preferably controlled to influence the degree of conjugation of themodifying groups to the peptide.

The present invention provides conditions under which the yield of apeptide, having the desired level of conjugation, is maximized. Theconditions in the exemplary embodiments of the inventions also recognizethe expense of the various reagents and the materials and time necessaryto purify the product: the reaction conditions set forth herein areoptimized to provide excellent yields of the desired product, whileminimizing waste of costly reagents.

II. The Compositions of Matter/Peptide Conjugates

In a first aspect, the present invention provides a conjugate between amodified sugar and a peptide. The present invention also provides aconjugate between a modifying group and a peptide. A peptide conjugatecan have one of several forms. In an exemplary embodiment, a peptideconjugate can comprise a peptide and a modifying group linked to anamino acid of the peptide through a glycosyl linking group. In anotherexemplary embodiment, a peptide conjugate can comprise a peptide and amodifying group linked to a glycosyl reside of the peptide through aglycosyl linking group. In another exemplary embodiment, the peptideconjugate can comprise a peptide and a glycosyl linking group which isbound to both a glycopeptide carbohydrate and directly to an amino acidresidue of the peptide backbone. In yet another exemplary embodiment, apeptide conjugate can comprise a peptide and a modifying group linkeddirectly to an amino acid residue of the peptide. In this embodiment,the peptide conjugate may not comprise a glycosyl group. In any of theseembodiments, the peptide may or not be glycosylated.

The conjugates of the invention will typically correspond to the generalstructure:

in which the symbols a, b, c, d and s represent a positive, non-zerointeger; and t is either 0 or a positive integer. The “agent”, ormodifying group, can be a therapeutic agent, a bioactive agent, adetectable label, a polymeric modifying group such as a water-solublepolymer (e.g., PEG, m-PEG, PPG, and m-PPG) or the like. The “agent”, ormodifying group, can be a peptide, e.g., enzyme, antibody, antigen, etc.The linker can be any of a wide array of linking groups, infra.Alternatively, the linker may be a single bond or a “zero order linker.”

II.A. Peptide

The present invention should in no way be construed as limited to aparticular nucleic acid and amino acid sequences set forth herein. Useof peptides of other sequences that are mutated to increase or decreasea property or modify a structural feature of the peptide are within thescope of the invention. For example, mutant peptides of use in theinvention include those that are provided with additionalO-glycosylation sites or such sites at other positions. Moreover, mutantpeptides that include one or more N-glycosylation site are of use in theinvention. Preferably, the amino acid sequence is at least about 96%,97%, 98% or 99% homologous to the amino acid sequences set forth herein.

In an exemplary embodiment, the amino acid residue to which the glycosyllinking group is attached is a member selected from serine, threonineand asparagine. In another exemplary embodiment, the peptide has asequence of SEQ ID NO: 2. In another exemplary embodiment, the aminoacid residue is a member selected from Asn 145, Asn 322 and combinationsthereof. In another exemplary embodiment, the peptide is a bioactivepeptide.

In yet another exemplary embodiment, the modified sugar and/or PEGmoiety on the peptide conjugate is located on the light chain. In yetanother exemplary embodiment, the modified sugar and/or PEG moiety onthe peptide conjugate is predominantly on the heavy chain. In yetanother exemplary embodiment, in a population of peptide conjugates, thelight chains predominantly contain a modified sugar and/or PEG moiety.In yet another exemplary embodiment, in a population of peptideconjugates, the heavy chains predominantly contain a modified sugarand/or PEG moiety.

In another exemplary embodiment, the ratio of light chain: heavy chainfunctionalization in the population is about 33:66. In another exemplaryembodiment, the ratio of light chain: heavy chain functionalization inthe population is about 35:65. In another exemplary embodiment, theratio of light chain: heavy chain functionalization in the population isabout 40:60. In another exemplary embodiment, the ratio of light chain:heavy chain functionalization in the population is about 45:55. Inanother exemplary embodiment, the ratio is about 50:50. In anotherexemplary embodiment, the ratio is about 55:45. In another exemplaryembodiment, the ratio is about 60:40. In another exemplary embodiment,the ratio is about 65:35. In another exemplary embodiment, the ratio isabout 66:33. In another exemplary embodiment, the ratio is about 70:30.In another exemplary embodiment, the ratio is about 75:25. In anotherexemplary embodiment, the ratio is about 80:20. In another exemplaryembodiment, the ratio is about 85:15. In another exemplary embodiment,the ratio is about 90:10. In another exemplary embodiment, the ratio oflight chain: heavy chain functionalization in the population is greaterthan about 90:10.

Methods for the expression and to determine the activity of peptides arewell known in the art, and are described in, for example, U.S. Pat. No.4,784,950. Briefly, expression of a peptide, or variants thereof, can beaccomplished in a variety of both prokaryotic and eukaryotic systems,including E. coli, CHO cells, BHK cells, insect cells using abaculovirus expression system, all of which are well known in the art.

Assays for the activity of a peptide conjugate prepared according to themethods of the present invention can be accomplished using methods wellknown in the art. As a non-limiting example, Quick et al. (HemorragicDisease and Thrombosis, 2nd ed., Leat Febiger, Philadelphia, 1966),describes a one-stage clotting assay useful for determining thebiological activity of a peptide prepared according to the methods ofthe present invention.

The peptides used in the invention are not limited to peptide when themodifying group is:

In these cases, the peptide in the peptide conjugate is a memberselected from the peptides in FIG. 13. In these cases, the peptide inthe peptide conjugate is a member selected from Factor VII, Factor VIIa,Factor VIII, Factor IX, Factor X, Factor XI, a peptide which is a memberselected from erythropoietin, granulocyte colony stimulating factor(G-CSF), Granulocyte-Macrophage Colony Stimulating Factor(GM-CSF)interferon alpha, interferon beta, interferon gamma,α₁-antitrypsin (ATT, or α-1 protease inhibitor, glucocerebrosidase,Tissue-Type Plasminogen Activator (TPA), Interleukin-2 (IL-2),urokinase, human DNase, insulin, Hepatitis B surface protein (HbsAg),human growth hormone, TNF Receptor-IgG Fc region fusion protein(Enbrel™), anti-HER2 monoclonal antibody (Herceptin™), monoclonalantibody to Protein F of Respiratory Syncytial Virus (Synagis™),monoclonal antibody to TNF-α (Remicade™), monoclonal antibody toglycoprotein IIb/IIIa (Reopro™), monoclonal antibody to CD20 (Rituxan™),anti-thrombin III (AT III), human Chorionic Gonadotropin (hCG),alpha-galactosidase (Fabrazyme™), alpha-iduronidase (Aldurazyme™),follicle stimulating hormone, beta-glucosidase, anti-TNF-alphamonoclonal antibody (MLB 5075), glucagon-like peptide-1 (GLP-1),beta-glucosidase (MLB 5064), alpha-galactosidase A (MLB 5082) andfibroblast growth factor. In an exemplary embodiment, the peptide isother than EPO.

In an exemplary embodiment, the polymeric modifying group has astructure according to the following formulae:

The peptides used in the invention are also not limited when themodifying group is:

In an exemplary embodiment, A¹ and A² are each members selected from —OHand —OCH₃.

Exemplary polymeric modifying groups according to this embodimentinclude:

In an exemplary embodiment, in which the modifying group is a branchedwater-soluble polymer, such as those shown above, it is generallypreferred that the concentration of sialidase is about 1.5 to about 2.5U/L of reaction mixture. More preferably the amount of sialidase isabout 2 U/L.

In another exemplary embodiment, about 5 to about 9 grams of peptidesubstrate is contacted with the amounts of sialidase set forth above.

The modified sugar is present in the reaction mixture in an amount fromabout 1 gram to about 6 grams, preferably from about 3 grams to about 4grams. It is generally preferred to maintain the concentration of amodified sugar having a branched water-soluble polymer modifying moiety,e.g., the moiety shown above, at less than about 0.5 mM. In a preferredembodiment, the modifying group is a branched poly(ethylene glycol)having a molecular weight from about 20 KDa to about 60 KDa, morepreferably, from about 30 KDa to about 50 KDa, and even more preferablyabout 40 KDa. An exemplary modifying group having a molecular weight ofabout 40 KDa is one that is from about 35 KDa to about 45 KDa.

Regarding the glycosyltransferase concentration, in a presentlypreferred embodiment, using the modifying group set forth above, theratio of glycosyltransferase to peptide is about 40 μg/mL transferase toabout 200 μM peptide.

II.B. Modified Sugar

In an exemplary embodiment, the peptides of the invention are reactedwith a modified sugar, thus forming a peptide conjugate. A modifiedsugar comprises a “sugar donor moiety” as well as a “sugar transfermoiety”. The sugar donor moiety is any portion of the modified sugarthat will be attached to the peptide, either through a glycosyl moietyor amino acid moiety, as a conjugate of the invention. The sugar donormoiety includes those atoms that are chemically altered during theirconversion from the modified sugar to the glycosyl linking group of thepeptide conjugate. The sugar transfer moiety is any portion of themodified sugar that will be not be attached to the peptide as aconjugate of the invention. For example, a modified sugar of theinvention is the PEGylated sugar nucleotide, PEG-sialic acid CMP. ForPEG-sialic acid CMP, the sugar donor moiety, or PEG-sialyl donor moiety,comprises PEG-sialic acid while the sugar transfer moiety, or sialyltransfer moiety, comprises CMP.

In modified sugars of use in the invention, the saccharyl moiety ispreferably a saccharide, a deoxy-saccharide, an amino-saccharide, or anN-acyl saccharide. The term “saccharide” and its equivalents,“saccharyl,” “sugar,” and “glycosyl” refer to monomers, dimers,oligomers and polymers. The sugar moiety is also functionalized with amodifying group. The modifying group is conjugated to the saccharylmoiety, typically, through conjugation with an amine, sulfhydryl orhydroxyl, e.g., primary hydroxyl, moiety on the sugar. In an exemplaryembodiment, the modifying group is attached through an amine moiety onthe sugar, e.g., through an amide, a urethane or a urea that is formedthrough the reaction of the amine with a reactive derivative of themodifying group.

Any saccharyl moiety can be utilized as the sugar donor moiety of themodified sugar. The saccharyl moiety can be a known sugar, such asmannose, galactose or glucose, or a species having the stereochemistryof a known sugar. The general formulae of these modified sugars are:

Other saccharyl moieties that are useful in forming the compositions ofthe invention include, but are not limited to fucose and sialic acid, aswell as amino sugars such as glucosamine, galactosamine, mannosamine,the 5-amine analogue of sialic acid and the like. The saccharyl moietycan be a structure found in nature or it can be modified to provide asite for conjugating the modifying group. For example, in oneembodiment, the modified sugar provides a sialic acid derivative inwhich the 9-hydroxy moiety is replaced with an amine. The amine isreadily derivatized with an activated analogue of a selected modifyinggroup.

Examples of modified sugars of use in the invention are described in PCTPatent Application No. PCT/US05/002522, which is herein incorporated byreference.

In a further exemplary embodiment, the invention utilizes modifiedsugars in which the 6-hydroxyl position is converted to thecorresponding amine moiety, which bears a linker-modifying groupcassette such as those set forth above. Exemplary glycosyl groups thatcan be used as the core of these modified sugars include Gal, GalNAc,Glc, GlcNAc, Fuc, Xyl, Man, and the like. A representative modifiedsugar according to this embodiment has the formula:

in which R¹¹-R¹⁴ are members independently selected from H, OH, C(O)CH₃,NH, and NH C(O)CH₃. R¹⁰ is a link to another glycosyl residue(—O-glycosyl) or to an amino acid of the peptide (—NH-(peptide)). R¹⁴ isOR¹, NHR¹ or NH-L-R¹. R¹ and NH-L-R¹ are as described above.

II.C. Glycosyl Linking Groups

In an exemplary embodiment, the invention provides a peptide conjugateformed between a modified sugar of the invention and a peptide. Inanother exemplary embodiment, when the modifying group on the modifiedsugar is

the peptide in the peptide conjugate is a member selected from thepeptides in FIG. 13. In yet another exemplary embodiment, the peptide inthe peptide conjugate is a member selected from Factor VII, Factor VIIa,Factor VIII, Factor IX, Factor X, Factor XI, erythropoietin, granulocytecolony stimulating factor (G-CSF), Granulocyte-Macrophage ColonyStimulating Factor (GM-CSF), interferon alpha, interferon beta,interferon gamma, α₁-antitrypsin (ATT, or α-1 protease inhibitor,glucocerebrosidase, Tissue-Type Plasminogen Activator (TPA),Interleukin-2 (IL-2), urokinase, human DNase, insulin, Hepatitis Bsurface protein (HbsAg), human growth hormone, TNF Receptor-IgG Fcregion fusion protein (Enbrel™), anti-HER2 monoclonal antibody(Herceptin™), monoclonal antibody to Protein F of Respiratory SyncytialVirus (Synagis™), monoclonal antibody to TNF-α (Remicade™), monoclonalantibody to glycoprotein IIb/IIIa (Reopro™), monoclonal antibody to CD20(Rituxan™), anti-thrombin III (AT III), human Chorionic Gonadotropin(hCG), alpha-galactosidase (Fabrazyme™), alpha-iduronidase(Aldurazyme™), follicle stimulating hormone, beta-glucosidase,anti-TNF-alpha monoclonal antibody (MLB 5075), glucagon-like peptide-1(GLP-1), beta-glucosidase (MLB 5064), alpha-galactosidase A (MLB 5082)and fibroblast growth factor. In this embodiment, the sugar donor moiety(such as the saccharyl moiety and the modifying group) of the modifiedsugar becomes a “glycosyl linking group”. The “glycosyl linking group”can alternatively refer to the glycosyl moiety which is interposedbetween the peptide and the modifying group.

In an exemplary embodiment, the polymeric modifying group has astructure according to the following formulae:

In an exemplary embodiment, modifying group on the modified sugar is:

In an exemplary embodiment, A¹ and A² are each members selected from —OHand —OCH₃.

Exemplary polymeric modifying groups according to this embodimentinclude:

Due to the versatility of the methods available for adding and/ormodifying glycosyl residues on a peptide, the glycosyl linking groupscan have substantially any structure. In the discussion that follows,the invention is illustrated by reference to the use of selectedderivatives of furanose and pyranose. Those of skill in the art willrecognize that the focus of the discussion is for clarity ofillustration and that the structures and compositions set forth aregenerally applicable across the genus of glycosyl linking groups andmodified sugars. The glycosyl linking group can comprise virtually anymono- or oligo-saccharide. The glycosyl linking groups can be attachedto an amino acid either through the side chain or through the peptidebackbone. Alternatively the glycosyl linking groups can be attached tothe peptide through a saccharyl moiety. This saccharyl moiety can be aportion of an O-linked or N-linked glycan structure on the peptide.

In an exemplary embodiment, the invention provides a peptide conjugatecomprising an intact glycosyl linking group having a formula that isselected from:

In Formulae I R² is H, CH₂OR⁷, COOR⁷ or OR⁷, in which R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl. When COOR⁷ is a carboxylic acid or carboxylate, both formsare represented by the designation of the single structure COO⁻ or COOH.In Formulae I and II, the symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR⁸,NHC(O)R⁹. The index d is 0 or 1. R⁸ and R⁹ are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, sialic acid or polysialic acid. At least one of R³, R⁴, R⁵,R⁶ or R^(6′) includes a modifying group. This modifying group can be apolymeric modifying moiety e.g., PEG, linked through a bond or a linkinggroup. In an exemplary embodiment, R⁶ and R^(6′), together with thecarbon to which they are attached are components of the pyruvyl sidechain of sialic acid. In a further exemplary embodiment, the pyruvylside chain is functionalized with the polymeric modifying group. Inanother exemplary embodiment, R⁶ and R^(6′), together with the carbon towhich they are attached are components of the side chain of sialic acidand the polymeric modifying group is a component of R⁵.

In an exemplary embodiment, the invention utilizes a glycosyl linkinggroup that has the formula:

in which J is a glycosyl moiety, L is a bond or a linker and R¹ is amodifying group, e.g., a polymeric modifying group. Exemplary bonds arethose that are formed between an NH₂ moiety on the glycosyl moiety and agroup of complementary reactivity on the modifying group. For example,when R¹ includes a carboxylic acid moiety, this moiety may be activatedand coupled with the NH₂ moiety on the glycosyl residue affording a bondhaving the structure NHC(O)R¹. J is preferably a glycosyl moiety that is“intact”, not having been degraded by exposure to conditions that cleavethe pyranose or furanose structure, e.g. oxidative conditions, e.g.,sodium periodate.

Exemplary linkers include alkyl and heteroalkyl moieties. The linkersinclude linking groups, for example acyl-based linking groups, e.g.,—C(O)NH—, —OC(O)NH—, and the like. The linking groups are bonds formedbetween components of the species of the invention, e.g., between theglycosyl moiety and the linker (L), or between the linker and themodifying group (R¹). Other exemplary linking groups are ethers,thioethers and amines. For example, in one embodiment, the linker is anamino acid residue, such as a glycine residue. The carboxylic acidmoiety of the glycine is converted to the corresponding amide byreaction with an amine on the glycosyl residue, and the amine of theglycine is converted to the corresponding amide or urethane by reactionwith an activated carboxylic acid or carbonate of the modifying group.

An exemplary species of NH-L-R¹ has the formula:—NH{C(O)(CH₂)_(a)NH}_(s){C(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NH}_(t)R¹,in which the indices s and t are independently 0 or 1. The indices a, band d are independently integers from 0 to 20, and c is an integer from1 to 2500. Other similar linkers are based on species in which an —NHmoiety is replaced by another group, for example, —S, —O or —CH₂. Asthose of skill will appreciate one or more of the bracketed moietiescorresponding to indices s and t can be replaced with a substituted orunsubstituted alkyl or heteroalkyl moiety.

More particularly, the invention utilizes compounds in which NH-L-R¹ is:NHC(O)(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)O(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NH(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)O(CH₂)_(d)NHR¹,NHC(O)(CH₂)_(a)NHR¹, NH(CH₂)_(a)NHR¹, and NHR¹. In these formulae, theindices a, b and d are independently selected from the integers from 0to 20, preferably from 1 to 5. The index c is an integer from 1 to about2500.

In an exemplary embodiment, c is selected such that the PEG moiety isapproximately 1 kD, 5 kD, 10, kD, 15 kD, 20 kD, 25 kD, 30 kD, 35 kD, 40kD or 45 kD.

For the purposes of convenience, the glycosyl linking groups in theremainder of this section will be based on a sialyl moiety. However, oneof skill in the art will recognize that another glycosyl moiety, such asmannosyl, galactosyl, glucosyl, or fucosyl, could be used in place ofthe sialyl moiety.

In an exemplary embodiment, the glycosyl linking group is an intactglycosyl linking group, in which the glycosyl moiety or moieties fanningthe linking group are not degraded by chemical (e.g., sodiummetaperiodate) or enzymatic (e.g., oxidase) processes. Selectedconjugates of the invention include a modifying group that is attachedto the amine moiety of an amino-saccharide, e.g., mannosamine,glucosamine, galactosamine, sialic acid etc. Exemplary modifyinggroup-intact glycosyl linking group cassettes according to this motifare based on a sialic acid structure, such as those having the formulae:

In the formulae above, R¹ and L are as described above. Further detailabout the structure of exemplary R¹ groups is provided below.

In still a further exemplary embodiment, the conjugate is formed betweena peptide and a modified sugar in which the modifying group is attachedthrough a linker at the 6-carbon position of the modified sugar. Thus,illustrative glycosyl linking groups according to this embodiment havethe formula:

in which the radicals are as discussed above. Glycosyl linking groupsinclude, without limitation, glucose, glucosamine, N-acetyl-glucosamine,galactose, galactosamine, N-acetyl-galactosamine, mannose, mannosamine,N-acetyl-mannosamine, and the like.

In one embodiment, the present invention provides a peptide conjugatecomprising the following glycosyl linking group:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched polyethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In one embodiment, the present invention provides a peptide conjugatecomprising the following glycosyl linking group:

D is a member selected from —OH and R¹-L-HN—; G is a member selectedfrom R¹-L- and —C(O)(C₁-C₆)alkyl-R¹; R¹ is a moiety comprising a memberselected from a straight-chain poly(ethylene glycol) residue andbranched poly(ethylene glycol) residue; and M is a member selected fromH, a salt counterion and a single negative charge; L is a linker whichis a member selected from a bond, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. In an exemplary embodiment,when D is OH, G is R¹-L-NH—. In another exemplary embodiment, when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In any the compounds of the invention, a COOH group can alternatively beCOOM, wherein M is a member selected from H, a negative charge, and asalt counterion.

The invention provides a peptide conjugate that includes a glycosyllinking group having the formula

In other embodiments, the glycosyl linking group has the formula:

in which the index t is 0 or 1.

In a still further exemplary embodiment, the glycosyl linking group hasthe formula:

in which the index t is 0 or 1.

In yet another embodiment, the glycosyl linking group has the formula:

in which the index p represents and integer from 1 to 10; and a iseither 0 or 1.

In another exemplary embodiment, the peptide conjugate comprises aglycosyl moiety selected from the formulae:

in which the index a and the linker L^(a) are as discussed above. Theindex p is an integer from 1 to 10. The indices t and a areindependently selected from 0 or 1. Each of these groups can be includedas components of the mono-, bi-, tri- and tetra-antennary saccharidestructures set forth above. AA is an amino acid residue of the peptide.

In an exemplary embodiment, the PEG moiety has a molecular weight ofabout 20 KDa. In another exemplary embodiment, the PEG moiety has amolecular weight of about 5 KDa. In another exemplary embodiment, thePEG moiety has a molecular weight of about 10 KDa. In another exemplaryembodiment, the PEG moiety has a molecular weight of about 40 KDa.

In an exemplary embodiment, the glycosyl linking group is a branchedSA-PEG-10 KDa moiety based on a cysteine residue, and one or two ofthese glycosyl linking groups are covalently attached to the peptide. Inanother exemplary embodiment, the glycosyl linking group is a branchedSA-PEG-10 KDa moiety based on a lysine residue, and one or two of theseglycosyl linking groups are covalently attached to the peptide. In anexemplary embodiment, the glycosyl linking group is a branched SA-PEG-10KDa moiety based on a cysteine residue, and one or two of these glycosyllinking groups are covalently attached to the peptide. In an exemplaryembodiment, the glycosyl linking group is a branched SA-PEG-10 KDamoiety based on a lysine residue, and one or two of these glycosyllinking groups are covalently attached to the peptide. In an exemplaryembodiment, the glycosyl linking group is a branched SA-PEG-5 KDa moietybased on a cysteine residue, and one, two or three of these glycosyllinking groups are covalently attached to the peptide. In an exemplaryembodiment, the glycosyl linking group is a branched SA-PEG-5 KDa moietybased on a lysine residue, and one, two or three of these glycosyllinking groups are covalently attached to the peptide. In an exemplaryembodiment, the glycosyl linking group is a branched SA-PEG-40 KDamoiety based on a cysteine residue, and one or two of these glycosyllinking groups are covalently attached to the peptide. In an exemplaryembodiment, the glycosyl linking group is a branched SA-PEG-40 KDamoiety based on a lysine residue, and one or two of these glycosyllinking groups are covalently attached to the peptide.

In an exemplary embodiment, a glycoPEGylated peptide conjugate of theinvention selected from the formulae set forth below:

In the formulae above, the index t is an integer from 0 to 1 and theindex p is an integer from 1 to 10. The symbol R^(15′) represents H, OH(e.g., Gal-OH), a sialyl moiety, a sialyl linking group (i.e., sialyllinking group-polymeric modifying group (Sia-L-R¹), or a sialyl moietyto which is bound a polymer modified sialyl moiety (e.g., Sia-Sia-L-R¹)(“Sia-Sia^(p)”)). Exemplary polymer modified saccharyl moieties have astructure according to Formulae I and II. An exemplary peptide conjugateof the invention will include at least one glycan having a R^(15′) thatincludes a structure according to Formulae I or II. The oxygen, with theopen valence, of Formulae I and II is preferably attached through aglycosidic linkage to a carbon of a Gal or GalNAc moiety. In a furtherexemplary embodiment, the oxygen is attached to the carbon at position 3of a galactose residue. In an exemplary embodiment, the modified sialicacid is linked α2,3-to the galactose residue. In another exemplaryembodiment, the sialic acid is linked α2,6-to the galactose residue.

In an exemplary embodiment, the sialyl linking group is a sialyl moietyto which is bound a polymer modified sialyl moiety (e.g., Sia-Sia-L-R¹)(“Sia-Sia^(p)”). Here, the glycosyl linking group is linked to agalactosyl moiety through a sialyl moiety:

An exemplary species according to this motif is prepared by conjugatingSia-L-R¹ to a terminal sialic acid of a glycan using an enzyme thatforms Sia-Sia bonds, e.g., CST-II, ST8Sia-II, ST8Sia-III and ST8Sia-IV.

In another exemplary embodiment, the glycans on the peptide conjugateshave a formula that is selected from the group:

and combinations thereof.

In each of the formulae above, R^(15′) is as discussed above. Moreover,an exemplary peptide conjugate of the invention will include at leastone glycan with an R¹⁵ moiety having a structure according to Formulae Ior II.

In another exemplary embodiment, the glycosyl linking group comprises atleast one glycosyl linking group having the formula:

wherein R¹⁵ is said sialyl linking group; and the index p is an integerselected from 1 to 10.

In an exemplary embodiment, the glycosyl linking moiety has the formula:

in which b is an integer from 0 to 1. The index s represents an integerfrom 1 to 10; and the index f represents an integer from 1 to 2500.

In an exemplary embodiment, the polymeric modifying group is PEG. Inanother exemplary embodiment, the PEG moiety has a molecular weight ofabout 20 KDa. In another exemplary embodiment, the PEG moiety has amolecular weight of about 5 KDa. In another exemplary embodiment, thePEG moiety has a molecular weight of about 10 KDa. In another exemplaryembodiment, the PEG moiety has a molecular weight of about 40 kDa. Inanother exemplary embodiment the glycosyl linking group is attached toAsn145, Asn322, Ser52, Ser60 or combinations thereof.

In an exemplary embodiment, the glycosyl linking group is a linearSA-PEG-10 KDa moiety, and one or two of these glycosyl linking groupsare covalently attached to the peptide. In another exemplary embodiment,the glycosyl linking group is a linear SA-PEG-20 KDa moiety, and one ortwo of these glycosyl linking groups are covalently attached to thepeptide. In an exemplary embodiment, the glycosyl linking group is alinear SA-PEG-5 KDa moiety, and one, two or three of these glycosyllinking groups are covalently attached to the peptide. In an exemplaryembodiment, the glycosyl linking group is a linear SA-PEG-40 KDa moiety,and one or two of these glycosyl linking groups are covalently attachedto the peptide.

In another exemplary embodiment, the glycosyl linking group is a sialyllinking group having the formula:

In another exemplary embodiment, Q is a member selected from H and CH₃.In another exemplary embodiment, wherein said glycosyl linking group hasthe formula:

wherein R¹⁵ is said sialyl linking group; and the index p is an integerselected from 1 to 10. In an exemplary embodiment, the glycosyl linkinggroup comprises the formula:

wherein the index b is an integer selected from 0 and 1. In an exemplaryembodiment, the index s is 1; and the index f is an integer selectedfrom about 200 to about 300. In another exemplary embodiment, theglycosyl linking group is a member selected from SA-PEG-10 KDa andSA-PEG-20 KDa, and wherein the number of said glycosyl linking groupswhich are covalently attached to the peptide is an integer selected from1 to 2. In another exemplary embodiment, the glycosyl linking group ismember selected from SA-PEG-5 KDa and SA-PEG-40 KDa, and wherein thenumber of said glycosyl linking groups which are covalently attached tothe peptide is an integer selected from 1 to 3.

II.D. Modifying Groups

The peptide conjugates of the invention comprise a modifying group. Thisgroup can be covalently attached to a peptide through an amino acid or aglycosyl linking group. In another exemplary embodiment, when themodifying group is

the peptide in the peptide conjugate is a member selected from thepeptides in FIG. 13. In another exemplary embodiment, the peptide in thepeptide conjugate is a member selected from Factor VII, Factor VIIa,Factor VIII, Factor IX, Factor X, Factor XI, erythropoietin, granulocytecolony stimulating factor (G-CSF), Granulocyte-Macrophage ColonyStimulating Factor (GM-CSF)interferon alpha, interferon beta, interferongamma, α₁-antitrypsin (ATT, or α-1 protease inhibitor,glucocerebrosidase, Tissue-Type Plasminogen Activator (TPA),Interleukin-2 (IL-2), urokinase, human DNase, insulin, Hepatitis Bsurface protein (HbsAg), human growth hormone, TNF Receptor-IgG Fcregion fusion protein (Enbrel™), anti-HER2 monoclonal antibody(Herceptin™), monoclonal antibody to Protein F of Respiratory SyncytialVirus (Synagis™), monoclonal antibody to TNF-α (Remicade™), monoclonalantibody to glycoprotein IIb/IIIa (Reopro™), monoclonal antibody to CD20(Rituxan™), anti-thrombin III (AT III), human Chorionic Gonadotropin(hCG), alpha-galactosidase (Fabrazyme™), alpha-iduronidase(Aldurazyme™), follicle stimulating hormone, beta-glucosidase,anti-TNF-alpha monoclonal antibody (MLB 5075), glucagon-like peptide-1(GLP-1), beta-glucosidase (MLB 5064), alpha-galactosidase A (MLB 5082)and fibroblast growth factor. “Modifying groups” can encompass a varietyof structures including targeting moieties, therapeutic moieties,biomolecules. Additionally, “modifying groups” include polymericmodifying groups, which are polymers which can alter a property of thepeptide such as its bioavailability or its half-life in the body.

In an exemplary embodiment, the polymeric modifying group has astructure according to the following formulae:

In another exemplary embodiment according to the formula above, thepolymeric modifying group has a structure according to the followingformula:

In an exemplary embodiment, A¹ and A² are each members selected from —OHand —OCH₃.

Exemplary polymeric modifying groups according to this embodimentinclude:

For the purposes of convenience, the modifying groups in the remainderof this section will be largely based on polymeric modifying groups suchas water soluble and water insoluble polymers. However, one of skill inthe art will recognize that other modifying groups, such as targetingmoieties, therapeutic moieties and biomolecules, could be used in placeof the polymeric modifying groups.

II.D.i. Linkers of the Modifying Groups

The linkers of the modifying group serve to attach the modifying group(ie polymeric modifying groups, targeting moieties, therapeutic moietiesand biomolecules) to the peptide. In an exemplary embodiment, thepolymeric modifying group is bound to a glycosyl linking group,generally through a heteroatom, e.g, nitrogen, on the core through alinker, L, as shown below:

R¹ is the polymeric moiety and L is selected from a bond and a linkinggroup. The index w represents an integer selected from 1-6, preferably1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety.

An exemplary compound according to the invention has a structureaccording to Formulae I or II above, in which at least one of R², R³,R⁴, R⁵, R⁶ or R^(6′) has the formula:

In another example according to this embodiment at least one of R², R³,R⁴, R⁵, R⁶ or R^(6′) has the formula:

in which s is an integer from 0 to 20 and R¹ is a linear polymericmodifying moiety.

In an exemplary embodiment, the polymeric modifying group-linkerconstruct is a branched structure that includes two or more polymericchains attached to central moiety. In this embodiment, the construct hasthe formula:

in which R¹ and L are as discussed above and w′ is an integer from 2 to6, preferably from 2 to 4 and more preferably from 2 to 3.

When L is a bond it is formed between a reactive functional group on aprecursor of R¹ and a reactive functional group of complementaryreactivity on the saccharyl core. When L is a non-zero order linker, aprecursor of L can be in place on the glycosyl moiety prior to reactionwith the R¹ precursor. Alternatively, the precursors of R¹ and L can beincorporated into a preformed cassette that is subsequently attached tothe glycosyl moiety. As set forth herein, the selection and preparationof precursors with appropriate reactive functional groups is within theability of those skilled in the art. Moreover, coupling the precursorsproceeds by chemistry that is well understood in the art.

In an exemplary embodiment, L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying group is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. The PEG moiety can be attached to the aminemoiety of the linker through an amide or urethane bond. The PEG islinked to the sulfur or oxygen atoms of cysteine and serine throughthioether or ether bonds, respectively.

In an exemplary embodiment, R⁵ includes the polymeric modifying group.In another exemplary embodiment, R⁵ includes both the polymericmodifying group and a linker, L, joining the modifying group to theremainder of the molecule. As discussed above, L can be a linear orbranched structure. Similarly, the polymeric modifying group can bebranched or linear.

II.D.ii. Water-Soluble Polymers

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.); poly(amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);nucleic acids; synthetic polymers (e.g., poly(acrylic acid),poly(ethers), e.g., poly(ethylene glycol); peptides, proteins, and thelike. The present invention may be practiced with any water-solublepolymer with the sole limitation that the polymer must include a pointat which the remainder of the conjugate can be attached.

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

Exemplary water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

The present invention is further illustrated by reference to apoly(ethylene glycol) conjugate. Several reviews and monographs on thefunctionalization and conjugation of PEG are available. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002). Routes for preparing reactive PEG molecules and formingconjugates using the reactive molecules are known in the art. Forexample, U.S. Pat. No. 5,672,662 discloses a water soluble andisolatable conjugate of an active ester of a polymer acid selected fromlinear or branched poly(alkylene oxides), poly(oxyethylated polyols),poly(olefinic alcohols), and poly(acrylomorpholine).

U.S. Pat. No. 6,376,604 sets forth a method for preparing awater-soluble 1-benzotriazolylcarbonate ester of a water-soluble andnon-peptidic polymer by reacting a terminal hydroxyl of the polymer withdi(1-benzotriazoyl)carbonate in an organic solvent. The active ester isused to form conjugates with a biologically active agent such as aprotein or peptide.

WO 99/45964 describes a conjugate comprising a biologically active agentand an activated water soluble polymer comprising a polymer backbonehaving at least one teiminus linked to the polymer backbone through astable linkage, wherein at least one terminus comprises a branchingmoiety having proximal reactive groups linked to the branching moiety,in which the biologically active agent is linked to at least one of theproximal reactive groups. Other branched poly(ethylene glycols) aredescribed in WO 96/21469, U.S. Pat. No. 5,932,462 describes a conjugateformed with a branched PEG molecule that includes a branched terminusthat includes reactive functional groups. The free reactive groups areavailable to react with a biologically active species, such as a proteinor peptide, forming conjugates between the poly(ethylene glycol) and thebiologically active species. U.S. Pat. No. 5,446,090 describes abifunctional PEG linker and its use in forming conjugates having apeptide at each of the PEG linker termini.

Conjugates that include degradable PEG linkages are described in WO99/34833; and WO 99/14259, as well as in U.S. Pat. No. 6,348,558. Suchdegradable linkages are applicable in the present invention.

The art-recognized methods of polymer activation set forth above are ofuse in the context of the present invention in the formation of thebranched polymers set forth herein and also for the conjugation of thesebranched polymers to other species, e.g., sugars, sugar nucleotides andthe like.

An exemplary water-soluble polymer is poly(ethylene glycol), e.g.,methoxy-poly(ethylene glycol). The poly(ethylene glycol) used in thepresent invention is not restricted to any particular form or molecularweight range. For unbranched poly(ethylene glycol) molecules themolecular weight is preferably between 500 and 100,000. A molecularweight of 2000-60,000 is preferably used and preferably of from about5,000 to about 40,000.

II.D.iii. Branched Water Soluble Polymers

In another embodiment the poly(ethylene glycol) is a branched PEG havingmore than one PEG moiety attached. Examples of branched PEGs aredescribed in U.S. Pat. No. 5,932,462; U.S. Pat. No. 5,342,940; U.S. Pat.No. 5,643,575; U.S. Pat. No. 5,919,455; U.S. Pat. No. 6,113,906; U.S.Pat. No. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5:283-288 (1994); and Yamasaki et al., Agric. Biol. Chem., 52: 2125-2127,1998. In a preferred embodiment the molecular weight of eachpoly(ethylene glycol) of the branched PEG is less than or equal to40,000 daltons.

Representative polymeric modifying moieties include structures that arebased on side chain-containing amino acids, e.g., serine, cysteine,lysine, and small peptides, e.g., lys-lys. Exemplary structures include:

Those of skill will appreciate that the free amine in the di-lysinestructures can also be pegylated through an amide or urethane bond witha PEG moiety.

In yet another embodiment, the polymeric modifying moiety is a branchedPEG moiety that is based upon a tri-lysine peptide. The tri-lysine canbe mono-, di-, tri-, or tetra-PEG-ylated. Exemplary species according tothis embodiment have the formulae:

in which the indices e, f and f′ are independently selected integersfrom 1 to 2500; and the indices q, q′ and q″ are independently selectedintegers from 1 to 20.

As will be apparent to those of skill, the branched polymers of use inthe invention include variations on the themes set forth above. Forexample the di-lysine-PEG conjugate shown above can include threepolymeric subunits, the third bonded to the α-amine shown as unmodifiedin the structure above. Similarly, the use of a tri-lysinefunctionalized with three or four polymeric subunits labeled with thepolymeric modifying moiety in a desired manner is within the scope ofthe invention.

As discussed herein, the PEG of use in the conjugates of the inventioncan be linear or branched. An exemplary precursor of use to form thebranched PEG containing peptide conjugates according to this embodimentof the invention has the formula:

Another exemplary precursor of use to form the branched PEG containingpeptide conjugates according to this embodiment of the invention has theformula:

The branched polymer species according to this formula are essentiallypure water-soluble polymers. X^(3′) is a moiety that includes anionizable (e.g., OH, COOH, H₂PO₄, HSO₃, HPO₃, and salts thereof, etc.)or other reactive functional group, e.g., infra. C is carbon. X⁵, R¹⁶and R¹⁷ are independently selected from non-reactive groups (e.g., H,unsubstituted alkyl, unsubstituted heteroalkyl) and polymeric arms(e.g., PEG). X² and X⁴ are linkage fragments that are preferablyessentially non-reactive under physiological conditions, which may bethe same or different. An exemplary linker includes neither aromatic norester moieties. Alternatively, these linkages can include one or moremoiety that is designed to degrade under physiologically relevantconditions, e.g., esters, disulfides, etc. X² and X⁴ join polymeric armsR¹⁶ and R¹⁷ to C. When X^(3′) is reacted with a reactive functionalgroup of complementary reactivity on a linker, sugar or linker-sugarcassette, X^(3′) is converted to a component of linkage fragment X³.

Exemplary linkage fragments for X², X³ and X⁴ are independently selectedand include S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH andNHC(O)O, and OC(O)NH, CH₂S, CH₂O, CH₂CH₂O, CH₂CH₂S, (CH₂)_(o),(CH₂)_(o)S or (CH₂)_(o)Y′-PEG wherein, Y′ is S, NH, NHC(O), C(O)NH,NHC(O)O, OC(O)NH, or O and o is an integer from 1 to 50. In an exemplaryembodiment, the linkage fragments X² and X⁴ are different linkagefragments.

In an exemplary embodiment, the precursor (Formula III), or an activatedderivative thereof, is reacted with, and thereby bound to a sugar, anactivated sugar or a sugar nucleotide through a reaction between X^(3′)and a group of complementary reactivity on the sugar moiety, e.g., anamine. Alternatively, X^(3′) reacts with a reactive functional group ona precursor to linker, L. One or more of R², R³, R⁴, R⁵, R⁶ or R^(6′) ofFormulae I and II can include the branched polymeric modifying moiety,or this moiety bound through L.

In an exemplary embodiment, the polymeric modifying group has astructure according to the following formulae:

In another exemplary embodiment according to the formula above, thebranched polymer has a structure according to the following formula:

In an exemplary embodiment, A¹ and A² are each selected from —OH and—OCH₃.

Exemplary polymeric modifying groups according to this embodimentinclude:

In an exemplary embodiment, the moiety:

is the linker arm, L. In this embodiment, an exemplary linker is derivedfrom a natural or unnatural amino acid, amino acid analogue or aminoacid mimetic, or a small peptide formed from one or more such species.For example, certain branched polymers found in the compounds of theinvention have the formula:

X^(a) is a linkage fragment that is formed by the reaction of a reactivefunctional group, e.g., X^(3′), on a precursor of the branched polymericmodifying moiety and a reactive functional group on the sugar moiety, ora precursor to a linker. For example, when X^(3′) is a carboxylic acid,it can be activated and bound directly to an amine group pendent from anamino-saccharide (e.g., Sia, GalNH₂, GlcNH₂, ManNH₂, etc.), forming aX^(a) that is an amide. Additional exemplary reactive functional groupsand activated precursors are described hereinbelow. The index crepresents an integer from 1 to 10. The other symbols have the sameidentity as those discussed above.

In another exemplary embodiment, X^(a) is a linking moiety formed withanother linker:

in which X^(b) is a second linkage fragment and is independentlyselected from those groups set forth for X^(a), and, similar to L, L¹ isa bond, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl.

Exemplary species for X^(a) and X^(b) include S, SC(O)NH, HNC(O)S,SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and OC(O)NH.

In another exemplary embodiment, X⁴ is a peptide bond to R¹⁷, which isan amino acid, di-peptide (e.g., Lys-Lys) or tri-peptide (e.g.,Lys-Lys-Lys) in which the alpha-amine moiety(ies) and/or side chainheteroatom(s) are modified with a polymeric modifying moiety.

In a further exemplary embodiment, the peptide conjugates of theinvention include a moiety, e.g., an R¹⁵ moiety that has a formula thatis selected from:

in which the identity of the radicals represented by the various symbolsis the same as that discussed hereinabove. L^(a) is a bond or a linkeras discussed above for L and L¹, e.g., substituted or unsubstitutedalkyl or substituted or unsubstituted heteroalkyl moiety. In anexemplary embodiment, L^(a) is a moiety of the side chain of sialic acidthat is functionalized with the polymeric modifying moiety as shown.Exemplary L^(a) moieties include substituted or unsubstituted alkylchains that include one or more OH or NH₂.

In yet another exemplary embodiment, the invention provides peptideconjugates having a moiety, e.g., an R¹⁵ moiety with formula:

The identity of the radicals represented by the various symbols is thesame as that discussed hereinabove. As those of skill will appreciate,the linker arm in Formulae VI and VII is equally applicable to othermodified sugars set forth herein. In exemplary embodiment, the speciesof Formulae VI and VII are the R¹⁵ moieties attached to the glycanstructures set forth herein.

In yet another exemplary embodiment, the peptide conjugate includes aR¹⁵ moiety with a formula which is a member selected from:

in which the identities of the radicals are as discussed above. Anexemplary species for L^(a) is —(CH₂)_(J)C(O)NH(CH₂)_(h)C(O)NH—, inwhich the indices h and j are independently selected integers from 0 to10. A further exemplary species is —C(O)NH—. The indices m and n areintegers independently selected from 0 to 5000. A¹, A², A³, A⁴, A⁵, A⁶,A⁷, A⁸, A⁹, A¹⁰ and A¹¹ are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, —NA¹²A¹³, —OA¹² and —SiA¹²A¹³.A¹² and A¹³ are members independently selected from substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

The embodiments of the invention set forth above are further exemplifiedby reference to species in which the polymer is a water-soluble polymer,particularly polyethylene glycol) (“PEG”), e.g., methoxy-poly(ethyleneglycol). Those of skill will appreciate that the focus in the sectionsthat follow is for clarity of illustration and the various motifs setforth using PEG as an exemplary polymer are equally applicable tospecies in which a polymer other than PEG is utilized.

PEG of any molecular weight, e.g., 1 KDa, 2 KDa, 5 KDa, 10 KDa, 15 KDa,20 KDa, 25 KDa, 30 KDa, 35 KDa, 40 KDa and 45 KDa is of use in thepresent invention.

In an exemplary embodiment, the R¹⁵ moiety has a formula that is amember selected from the group:

In each of the structures above, the linker fragment —NH(CH₂)_(a)— canbe present or absent.

In other exemplary embodiments, the peptide conjugate includes an R¹⁵moiety selected from the group:

In each of the formulae above, the indices e and f are independentlyselected from the integers from 1 to 2500. In further exemplaryembodiments, e and f are selected to provide a PEG moiety that is about1 KDa, 2 KDa, 5 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa, 35 KDa, 40KDa and 45 KDa. The symbol Q represents substituted or unsubstitutedalkyl (e.g., C₁-C₆ alkyl, e.g., methyl), substituted or unsubstitutedheteroalkyl or H.

Other branched polymers have structures based on di-lysine (Lys-Lys)peptides, e.g.:

and tri-lysine peptides (Lys-Lys-Lys), e.g.:

In each of the figures above, the indices e, f, f′ and f″ representintegers independently selected from 1 to 2500. The indices q, q′ and q″represent integers independently selected from 1 to 20.

In another exemplary embodiment, the modifying group:

has a formula that is a member selected from:

wherein Q is a member selected from H and substituted or unsubstitutedC₁-C₆ alkyl. The indices e and f are integers independently selectedfrom 1 to 2500, and the index q is an integer selected from 0 to 20.

In another exemplary embodiment, the modifying group:

has a formula that is a member selected from:

wherein Q is a member selected from H and substituted or unsubstitutedC₁-C₆ alkyl. The indices e, f and f′ are integers independently selectedfrom 1 to 2500, and q and q′ are integers independently selected from 1to 20.

In another exemplary embodiment, the branched polymer has a structureaccording to the following formula:

in which the indices m and n are integers independently selected from 0to 5000. A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, A¹⁰ and A¹¹ are membersindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,—NA12A¹³, —OA¹² and —SiA¹²A¹³. A¹² and A¹³ and are members independentlyselected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl.

Formula IIIa is a subset of Formula III. The structures described byFormula IIIa are also encompassed by Formula III.

In an exemplary embodiment, the polymeric modifying group has astructure according to the following formulae:

In another exemplary embodiment according to the formula above, thebranched polymer has a structure according to the following formula:

In an exemplary embodiment, A¹ and A² are members independently selectedfrom —OH and —OCH₃.

Exemplary polymeric modifying groups according to this embodimentinclude:

In an illustrative embodiment, the modified sugar is sialic acid andselected modified sugar compounds of use in the invention have theformulae:

The indices a, b and d are integers from 0 to 20. The index c is aninteger from 1 to 2500. The structures set forth above can be componentsof R¹⁵.

In another illustrative embodiment, a primary hydroxyl moiety of thesugar is functionalized with the modifying group. For example, the9-hydroxyl of sialic acid can be converted to the corresponding amineand functionalized to provide a compound according to the invention.Formulae according to this embodiment include:

The structures set forth above can be components of R¹⁵.

Although the present invention is exemplified in the preceding sectionsby reference to PEG, as those of skill will appreciate, an array ofpolymeric modifying moieties is of use in the compounds and methods setforth herein.

In selected embodiments, R¹ or L-R¹ is a branched PEG, for example, oneof the species set forth above. In an exemplary embodiment, the branchedPEG structure is based on a cysteine peptide. Illustrative modifiedsugars according to this embodiment include:

in which X⁴ is a bond or O. In each of the structures above, thealkylamine linker —(CH₂)_(a)NH— can be present or absent. The structuresset forth above can be components of R¹⁵/R^(15′).

As discussed herein, the polymer-modified sialic acids of use in theinvention may also be linear structures. Thus, the invention providesfor conjugates that include a sialic acid moiety derived from astructure such as:

in which the indices q and e are as discussed above.

Exemplary modified sugars are modified with water-soluble orwater-insoluble polymers. Examples of useful polymer are furtherexemplified below.

In another exemplary embodiment, the peptide is derived from insectcells, remodeled by adding GlcNAc and Gal to the mannose core andglycopegylated using a sialic acid bearing a linear PEG moiety,affording a peptide that comprises at least one moiety having theformula:

in which the index t is an integer from 0 to 1; the index s representsan integer from 1 to 10; and the index f represents an integer from 1 to2500.

In one embodiment, the present invention provides a peptide conjugatecomprising the following glycosyl linking group:

D is a member selected from —OH and R¹-L-HN—; G is a member selectedfrom R¹-L- and —C(O)(C₁-C₆)alkyl-R¹; R¹ is a moiety comprising a memberselected from a straight-chain poly(ethylene glycol) residue andbranched poly(ethylene glycol) residue; and M is a member selected fromH, a salt counterion and a single negative charge; L is a linker whichis a member selected from a bond, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl. In an exemplary embodiment,when D is OH, G is R¹-L-. In another exemplary embodiment, when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In an exemplary embodiment, L-R¹ has the formula:

wherein a is an integer selected from 0 to 20.

In an exemplary embodiment, R¹ has a structure that is a member selectedfrom:

wherein e, f, m and n are integers independently selected from 1 to2500; and q is an integer selected from 0 to 20.

In an exemplary embodiment, R¹ has a structure that is a member selectedfrom:

wherein e, f and f′ are integers independently selected from 1 to 2500;and q and q′ are integers independently selected from 1 to 20.

In another exemplary embodiment, R¹ has a structure that is a memberselected from:

wherein e, f and f′ are integers independently selected from 1 to 2500;and q and q′ are integers independently selected from 1 to 20.

In another exemplary embodiment, R¹ has a structure that is a memberselected from:

wherein e and f are integers independently selected from 1 to 2500

In another exemplary embodiment, the glycosyl linker has the formula:

In another exemplary embodiment, the peptide conjugate comprises atleast one of said glycosyl linker according to a formula selected from:

wherein AA is an amino acid residue of said peptide conjugate and t isan integer selected from 0 and 1.

In another exemplary embodiment, the peptide conjugate comprises atleast one of said glycosyl linker wherein each of said glycosyl linkerhas a structure which is a member independently selected from thefollowing formulae:

wherein AA is an amino acid residue of said peptide conjugate and t isan integer selected from 0 and 1.

In another exemplary embodiment, the peptide conjugate comprises atleast one of said glycosyl linker according to a formula selected from:

wherein AA is an amino acid residue of said peptide conjugate and t isan integer selected from 0 and 1. In an exemplary embodiment, a memberselected from 0 and 2 of the sialyl moieties which do not comprise G areabsent. In an exemplary embodiment, a member selected from 1 and 2 ofthe sialyl moieties which do not comprise G are absent.

In another exemplary embodiment, the peptide conjugate comprises atleast one of said glycosyl linker according to a formula selected from:

wherein AA is an amino acid residue of said peptide conjugate and t isan integer selected from 0 and 1. In an exemplary embodiment, a memberselected from 0 and 2 of the sialyl moieties which do not comprise G areabsent. In an exemplary embodiment, a member selected from 1 and 2 ofthe sialyl moieties which do not comprise G are absent.

In another exemplary embodiment, the peptide conjugate comprises atleast one said glycosyl linker according to a formula selected from:

wherein AA is an amino acid residue of said peptide conjugate and t isan integer selected from 0 and 1. In an exemplary embodiment, a memberselected from 0 and 2 of the sialyl moieties which do not comprise G areabsent. In an exemplary embodiment, a member selected from 1 and 2 ofthe sialyl moieties which do not comprise G are absent.

In another exemplary embodiment, the peptide has the amino acid sequenceof SEQ ID NO: 2. In another exemplary embodiment, the glycosyl linker isattached to the peptide through an amino acid residue selected fromserine and threonine.

In another exemplary embodiment, the asparagine residue is a memberselected from N152, N322 and combinations thereof.

In another exemplary embodiment, the peptide is a bioactive peptide.

In another exemplary embodiment, the glycosyl linker is attached to saidpeptide through an amino acid residue which is an asparagine residue.

In another exemplary embodiment, the invention provides a peptide whichis produced in a suitable host. The invention also provides methods ofexpressing this peptide.

In another exemplary embodiment, the host is a mammalian expressionsystem.

In another exemplary embodiment, the invention provides a method oftreating a condition in a subject in need thereof, said conditioncharacterized by compromised clotting potency in said subject, saidmethod comprising the step of administering to the subject an amount ofthe peptide conjugate of invention, effective to ameliorate saidcondition in said subject. In another exemplary embodiment, the methodcomprises administering to said mammal an amount of the peptideconjugate produced according to the methods described herein.

In another aspect, the invention provides a method of making a peptideconjugate comprising a glycosyl linker comprising a modified sialylresidue having the formula:

wherein R² is H, CH₂OR⁷, COOR⁷ or OR⁷. R⁷ represents H, substituted orunsubstituted alkyl or substituted or unsubstituted heteroalkyl. R³ andR⁴ are members independently selected from H, substituted orunsubstituted alkyl, OR^(B), NHC(O)R⁹. R⁸ and R⁹ are independentlyselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl or sialic acid. R¹⁶ and R¹⁷ are independentlyselected polymeric arms. X² and X⁴ are independently selected linkagefragments joining polymeric moieties R¹⁶ and R¹⁷ to C. X⁵ is anon-reactive group and L^(a) is a linker group. The method comprises (a)contacting a peptide comprising the glycosyl moiety:

with a PEG-sialic acid donor moiety having the formula:

and an enzyme that transfers PEG-sialic acid onto the Gal of saidglycosyl moiety, under conditions appropriate for said transfer.

In another exemplary embodiment, the moiety:

has a formula that is a member selected from:

wherein e, f, m and n are integers independently selected from 1 to2500; and q is an integer selected from 0 to 20.

In another exemplary embodiment, the moiety:

has a formula that is a member selected from:

wherein e, f and f′ are integers independently selected from 1 to 2500;and q and q′ are integers independently selected from 1 to 20.

In another exemplary embodiment, the glycosyl linker comprises theformula:

In another exemplary embodiment, the peptide conjugate comprises atleast one glycosyl linker having the formula:

wherein AA is an amino acid residue of said peptide; t is an integerselected from 0 and 1; and R¹⁵ is the modified sialyl moiety.

In another exemplary embodiment, the peptide has the amino acid sequenceof SEQ ID NO: 2.

In another exemplary embodiment, the glycosyl linker is attached to saidpeptide through an amino acid residue which is an asparagine residue.

In another exemplary embodiment, the asparagine residue is a memberselected from N152, N322 and combinations thereof.

In another exemplary embodiment, the peptide is a bioactive peptide.

In another exemplary embodiment, the method comprises, prior to step(a): (b) expressing the peptide in a suitable host.

In another aspect, the invention provides a method of treating acondition in a subject in need thereof, said condition characterized bycompromised clotting potency in said subject, said method comprising thestep of administering to the subject an amount of the peptide conjugateproduced according to the methods described herein, effective toameliorate said condition in said subject. In another exemplaryembodiment, the method comprises administering to said mammal an amountof the peptide conjugate produced according to the methods describedherein.

In another aspect, the invention provides a method of synthesizing apeptide conjugate, said method comprising combining a) sialidase; b)enzyme which is a member selected from glycosyltransferase,exoglycosidase and endoglycosidase; c) modified sugar/modified sialylresidue; d) peptide thus synthesizing said peptide conjugate. In anexemplary embodiment, the combining is for a time less than 10 hours. Inanother exemplary embodiment, the invention further comprises a cappingstep.

II.D.iv. Water-Insoluble Polymers

In another embodiment, analogous to those discussed above, the modifiedsugars include a water-insoluble polymer, rather than a water-solublepolymer. The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic peptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

The motifs forth above for R¹, L-R¹, R¹⁵, R^(15′) and other radicals areequally applicable to water-insoluble polymers, which may beincorporated into the linear and branched structures without limitationutilizing chemistry readily accessible to those of skill in the art.

Representative water-insoluble polymers include, but are not limited to,polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

Synthetically modified natural polymers of use in conjugates of theinvention include, but are not limited to, alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, andnitrocelluloses. Particularly preferred members of the broad classes ofsynthetically modified natural polymers include, but are not limited to,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate,cellulose sulfate sodium salt, and polymers of acrylic and methacrylicesters and alginic acid.

These and the other polymers discussed herein can be readily obtainedfrom commercial sources such as Sigma Chemical Co. (St. Louis, Mo.),Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

Representative biodegradable polymers of use in the conjugates of theinvention include, but are not limited to, polylactides, polyglycolidesand copolymers thereof, poly(ethylene terephthalate), poly(butyricacid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

The polymers of use in the invention include “hybrid” polymers thatinclude water-insoluble materials having within at least a portion oftheir structure, a bioresorbable molecule. An example of such a polymeris one that includes a water-insoluble copolymer, which has abioresorbable region, a hydrophilic region and a plurality ofcrosslinkable functional groups per polymer chain.

For purposes of the present invention, “water-insoluble materials”includes materials that are substantially insoluble in water orwater-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thepolymer molecule, as a whole, does not to any substantial measuredissolve in water.

For purposes of the present invention, the term “bioresorbable molecule”includes a region that is capable of being metabolized or broken downand resorbed and/or eliminated through normal excretory routes by thebody. Such metabolites or break down products are preferablysubstantially non-toxic to the body.

The bioresorbable region may be either hydrophobic or hydrophilic, solong as the copolymer composition as a whole is not renderedwater-soluble. Thus, the bioresorbable region is selected based on thepreference that the polymer, as a whole, remains water-insoluble.Accordingly, the relative properties, i.e., the kinds of functionalgroups contained by, and the relative proportions of the bioresorbableregion, and the hydrophilic region are selected to ensure that usefulbioresorbable compositions remain water-insoluble.

Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.Biomed. Mater. Res. 22: 993-1009 (1988).

Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly (amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebiosresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

Higher order copolymers can also be used in the present invention. Forexample, Casey et al., U.S. Pat. No. 4,438,253, which issued on Mar. 20,1984, discloses tri-block copolymers produced from thetransesterification of poly(glycolic acid) and an hydroxyl-endedpoly(alkylene glycol). Such compositions are disclosed for use asresorbable monofilament sutures. The flexibility of such compositions iscontrolled by the incorporation of an aromatic orthocarbonate, such astetra-p-tolyl orthocarbonate into the copolymer structure.

Other polymers based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha di-functional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

Bioresorbable regions of coatings useful in the present invention can bedesigned to be hydrolytically and/or enzymatically cleavable. Forpurposes of the present invention, “hydrolytically cleavable” refers tothe susceptibility of the copolymer, especially the bioresorbableregion, to hydrolysis in water or a water-containing environment.Similarly, “enzymatically cleavable” as used herein refers to thesusceptibility of the copolymer, especially the bioresorbable region, tocleavage by endogenous or exogenous enzymes.

When placed within the body, the hydrophilic region can be processedinto excretable and/or metabolizable fragments. Thus, the hydrophilicregion can include, for example, polyethers, polyalkylene oxides,polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol), poly(alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins andcopolymers and mixtures thereof. Furthermore, the hydrophilic region canalso be, for example, a poly(alkylene)oxide. Such poly(alkylene)oxidescan include, for example, poly(ethylene)oxide, poly(propylene)oxide andmixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the presentinvention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

Bio-compatible hydrogel compositions whose integrity can be controlledthrough crosslinking are known and are presently preferred for use inthe methods of the invention. For example, Hubbell et al., U.S. Pat.Nos. 5,410,016, which issued on Apr. 25, 1995 and 5,529,914, whichissued on Jun. 25, 1996, disclose water-soluble systems, which arecrosslinked block copolymers having a water-soluble central blocksegment sandwiched between two hydrolytically labile extensions. Suchcopolymers are further end-capped with photopolymerizable acrylatefunctionalities. When crosslinked, these systems become hydrogels. Thewater soluble central block of such copolymers can include poly(ethyleneglycol); whereas, the hydrolytically labile extensions can be apoly(α-hydroxy acid), such as polyglycolic acid or polylactic acid. See,Sawhney et al., Macromolecules 26: 581-587 (1993).

In another preferred embodiment, the gel is a thermoreversible gel.Thermoreversible gels including components, such as pluronics, collagen,gelatin, hyalouronic acid, polysaccharides, polyurethane hydrogel,polyurethane-urea hydrogel and combinations thereof are presentlypreferred.

In yet another exemplary embodiment, the conjugate of the inventionincludes a component of a liposome. Liposomes can be prepared accordingto methods known to those skilled in the art, for example, as describedin Eppstein et al., U.S. Pat. No. 4,522,811. For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound or its pharmaceutically acceptable salt is then introduced intothe container. The container is then swirled by hand to free lipidmaterial from the sides of the container and to disperse lipidaggregates, thereby forming the liposomal suspension.

The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, is of use in thepresent invention.

The structural formats discussed above in the context of thewater-soluble polymers, both straight-chain and branched are generallyapplicable with respect to the water-insoluble polymers as well. Thus,for example, the cysteine, serine, dilysine, and trilysine branchingcores can be functionalized with two water-insoluble polymer moieties.The methods used to produce these species are generally closelyanalogous to those used to produce the water-soluble polymers.

II.D.v. Methods of Producing the Polymeric Modifying Groups

The polymeric modifying groups can be activated for reaction with aglycosyl or saccharyl moiety or an amino acid moiety. Exemplarystructures of activated species (e.g., carbonates and active esters)include:

In the figure above, q is a member selected from 1-40. Other activating,or leaving groups, appropriate for activating linear and branched PEGsof use in preparing the compounds set forth herein include, but are notlimited to the species:

PEG molecules that are activated with these and other species andmethods of making the activated PEGs are set forth in WO 04/083259.

Those of skill in the art will appreciate that one or more of the m-PEGarms of the branched polymers shown above can be replaced by a PEGmoiety with a different terminus, e.g., OH, COOH, NH₂, C₂-C₁₀-alkyl,etc. Moreover, the structures above are readily modified by insertingalkyl linkers (or removing carbon atoms) between the α-carbon atom andthe functional group of the amino acid side chain. Thus, “homo”derivatives and higher homologues, as well as lower homologues arewithin the scope of cores for branched PEGs of use in the presentinvention.

The branched PEG species set forth herein are readily prepared bymethods such as that set forth in the scheme below:

in which X^(d) is O or S and r is an integer from 1 to 5. The indices eand f are independently selected integers from 1 to 2500. In anexemplary embodiment, one or both of these indices are selected suchthat the polymer is about 5 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa,35 KDa, or 40 KDa in molecular weight.

Thus, according to this scheme, a natural or unnatural amino acid iscontacted with an activated m-PEG derivative, in this case the tosylate,forming 1 by alkylating the side-chain heteroatom X^(d). Themono-functionalize m-PEG amino acid is submitted to N-acylationconditions with a reactive m-PEG derivative, thereby assembling branchedm-PEG 2. As one of skill will appreciate, the tosylate leaving group canbe replaced with any suitable leaving group, e.g., halogen, mesylate,triflate, etc. Similarly, the reactive carbonate utilized to acylate theamine can be replaced with an active ester, e.g., N-hydroxysuccinimide,etc., or the acid can be activated in situ using a dehydrating agentsuch as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.

In other exemplary embodiments, the urea moiety is replaced by a groupsuch as a amide.

II.E. Homodisperse Peptide Conjugate Compositions of Matter

In addition to providing peptide conjugates that are formed through achemically or enzymatically added glycosyl linking group, the presentinvention provides compositions of matter comprising peptide conjugatesthat are highly homogenous in their substitution patterns. Using themethods of the invention, it is possible to form peptide conjugates inwhich substantial proportion of the glycosyl linking groups and glycosylmoieties across a population of peptide conjugates are attached to astructurally identical amino acid or glycosyl residue. Thus, in a secondaspect, the invention provides a peptide conjugate having a populationof water-soluble polymer moieties, which are covalently bound to thepeptide through a glycosyl linking group, e.g., an intact glycosyllinking group. In a an exemplary peptide conjugate of the invention,essentially each member of the water soluble polymer population is boundvia the glycosyl linking group to a glycosyl residue of the peptide, andeach glycosyl residue of the peptide to which the glycosyl linking groupis attached has the same structure.

The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a modifying group,e.g. therapeutic moiety, diagnostic moiety, targeting moiety, toxinmoiety or the like via a glycosyl linking group. Each of theabove-recited modifying groups can be a small molecule, natural polymer(e.g., polypeptide) or synthetic polymer. When the modifying group isattached to a sialic acid, it is generally preferred that the modifyinggroup is substantially non-fluorescent.

In an exemplary embodiment, the peptides of the invention include atleast one O-linked or N-linked glycosylation site, which is glycosylatedwith a modified sugar that includes a polymeric modifying group, e.g., aPEG moiety. In an exemplary embodiment, the PEG is covalently attachedto the peptide via an intact glycosyl linking group, or via anon-glycosyl linker, e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl. The glycosyl linking group iscovalently attached to either an amino acid residue or a glycosylresidue of the peptide. Alternatively, the glycosyl linking group isattached to one or more glycosyl units of a glycopeptide. The inventionalso provides conjugates in which a glycosyl linking group is attachedto both an amino acid residue and a glycosyl residue.

The glycans on the peptides of the invention generally correspond tothose found on a peptide that is produced by mammalian (BHK, CHO) cellsor insect (e.g., Sf-9) cells, following remodeling according to themethods set forth herein. For example, insect-derived peptide that isexpressed with a tri-mannosyl core is subsequently contacted with aGlcNAc donor and a GlcNAc transferase and a Gal donor and a Galtransferase. Appending GlcNAc and Gal to the tri-mannosyl core isaccomplished in either two steps or a single step. A modified sialicacid is added to at least one branch of the glycosyl moiety as discussedherein. Those Gal moieties that are not functionalized with the modifiedsialic acid are optionally “capped” by reaction with a sialic acid donorin the presence of a sialyl transferase.

In an exemplary embodiment, at least 60% of terminal Gal moieties in apopulation of peptides is capped with sialic acid, preferably at least70%, more preferably, at least 80%, still more preferably at least 90%and even more preferably at least 95%, 96%, 97%, 98% or 99% are cappedwith sialic acid.

II.F. Nucleotide Sugars

In another aspect of the invention, the invention also provides sugarnucleotides. Exemplary species according to this embodiment include:

in which the index y is an integer selected from 0, 1 and 2. Base is anucleic acid base, such as adenine, thymine, guanine, cytidine anduridine. R², R³ and R⁴ are as described above. In an exemplaryembodiment, L-(R¹)_(w) is a member selected from

in which the variables are as described above.

In an exemplary embodiment, L-(R¹)_(w) has a structure according to thefollowing formula:

In an exemplary embodiment, A¹ and A² are each selected from —OH and—OCH₃.

Exemplary polymeric modifying groups according to this embodimentinclude:

In another exemplary embodiment, the nucleotide sugars have a formulawhich is a member selected from:

An exemplary nucleotide sugar according to this embodiment has thestructure:

An exemplary nucleotide sugar according to this embodiment has thestructure:

In another exemplary embodiment, the nucleotide sugar is based upon thefollowing formula:

in which the R groups, and L, represent moieties as discussed above. Theindex “y” is 0, 1 or 2. In an exemplary embodiment, L is a bond betweenNH and R¹. The base is a nucleic acid base.

In an exemplary embodiment, L-R¹ is a member selected from

in which the variables are as described above.

In an exemplary embodiment, L-R¹ has a structure according to thefollowing formula:

In an exemplary embodiment, A¹ and A² are each sleeted from —OH and—OCH₃.

III. The Methods

In addition to the conjugates discussed above, the present inventionprovides methods for preparing these and other conjugates. Moreover, theinvention provides methods of preventing, curing or ameliorating adisease state by administering a conjugate of the invention to a subjectat risk of developing the disease or a subject that has the disease.

In exemplary embodiments, the conjugate is formed between a polymericmodifying moiety and a glycosylated or non-glycosylated peptide. Thepolymer is conjugated to the peptide via a glycosyl linking group, whichis interposed between, and covalently linked to both the peptide (orglycosyl residue) and the modifying group (e.g., water-soluble polymer).The method includes contacting the peptide with a mixture containing amodified sugar and an enzyme, e.g., a glycosyltransferase thatconjugates the modified sugar to the substrate. The reaction isconducted under conditions appropriate to form a covalent bond betweenthe modified sugar and the peptide. The sugar moiety of the modifiedsugar is preferably selected from nucleotide sugars. The method ofsynthesizing a peptide conjugate, comprising combining a) sialidase; b)an enzyme capable of catalyzing the transfer of a glycosyl linking groupsuch as a glycosyltransferase, exoglycosidase or endoglycosidase; c)modified sugar; d) a peptide, thus synthesizing the peptide conjugate.The reaction is conducted under conditions appropriate to form acovalent bond between the modified sugar and the peptide. The sugarmoiety of the modified sugar is preferably selected from nucleotidesugars.

In an exemplary embodiment, the modified sugar, such as those set forthabove, is activated as the corresponding nucleotide sugars. Exemplarysugar nucleotides that are used in the present invention in theirmodified form include nucleotide mono-, di- or triphosphates or analogsthereof. In a preferred embodiment, the modified sugar nucleotide isselected from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Evenmore preferably, the sugar nucleotide portion of the modified sugarnucleotide is selected from UDP-galactose, UDP-galactosamine,UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid,or CMP-NeuAc. In an exemplary embodiment, the nucleotide phosphate isattached to C-1.

The invention also provides for the use of sugar nucleotides modifiedwith L-R¹ at the 6-carbon position. Exemplary species according to thisembodiment include:

in which the R groups, and L, represent moieties as discussed above. Theindex “y” is 0, 1 or 2. In an exemplary embodiment, L is a bond betweenNH and R¹. The base is a nucleic acid base.

Exemplary nucleotide sugars of use in the invention in which the carbonat the 6-position is modified include species having the stereochemistryof GDP mannose, e.g.:

in which X⁵ is a bond or 0. The index i represents 0 or 1. The index arepresents an integer from 1 to 20. The indices e and f independentlyrepresent integers from 1 to 2500. Q, as discussed above, is H orsubstituted or unsubstituted C₁-C₆ alkyl. As those of skill willappreciate, the serine derivative, in which S is replaced with 0 alsofalls within this general motif.

In a still further exemplary embodiment, the invention provides aconjugate in which the modified sugar is based on the stereochemistry ofUDP galactose. An exemplary nucleotide sugar of use in this inventionhas the structure:

In another exemplary embodiment, the nucleotide sugar is based on thestereochemistry of glucose. Exemplary species according to thisembodiment have the formulae:

Thus, in an illustrative embodiment in which the glycosyl moiety issialic acid, the method of the invention utilizes compounds having theformulae:

in which L-R¹ is as discussed above, and L¹-R¹ represents a linker boundto the modifying group. As with L, exemplary linker species according toL¹ include a bond, alkyl or heteroalkyl moieties.

Moreover, as discussed above, the present invention provides for the useof nucleotide sugars that are modified with a water-soluble polymer,which is either straight-chain or branched. For example, compoundshaving the formula shown below are of use to prepare conjugates withinthe scope of the present invention:

in which X⁴ is 0 or a bond.

In general, the sugar moiety or sugar moiety-linker cassette and the PEGor PEG-linker cassette groups are linked together through the use ofreactive groups, which are typically transformed by the linking processinto a new organic functional group or unreactive species. The sugarreactive functional group(s), is located at any position on the sugarmoiety. Reactive groups and classes of reactions useful in practicingthe present invention are generally those that are well known in the artof bioconjugate chemistry. Currently favored classes of reactionsavailable with reactive sugar moieties are those, which proceed underrelatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

Useful reactive functional groups pendent from a sugar nucleus ormodifying group include, but are not limited to:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups, which can be converted to, e.g., esters,        ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the functional group of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc; and    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In the discussion that follows, a number of specific examples ofmodified sugars that are useful in practicing the present invention areset forth. In the exemplary embodiments, a sialic acid derivative isutilized as the sugar nucleus to which the modifying group is attached.The focus of the discussion on sialic acid derivatives is for clarity ofillustration only and should not be construed to limit the scope of theinvention. Those of skill in the art will appreciate that a variety ofother sugar moieties can be activated and derivatized in a manneranalogous to that set forth using sialic acid as an example. Forexample, numerous methods are available for modifying galactose,glucose, N-acetylgalactosamine and fucose to name a few sugarsubstrates, which are readily modified by art recognized methods. See,for example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schaferet al., J. Org. Chem. 65: 24 (2000)).

In an exemplary embodiment, the modified sugar is based upon a6-amino-N-acetyl-glycosyl moiety.

In the scheme above, the index n represents an integer from 1 to 2500.In an exemplary embodiment, this index is selected such that the polymeris about 10 KDa, 15 KDa or 20 KDa in molecular weight. The symbol “A”represents an activating group, e.g., a halo, a component of anactivated ester (e.g., a N-hydroxysuccinimide ester), a component of acarbonate (e.g., p-nitrophenyl carbonate) and the like. Those of skillin the art will appreciate that other PEG-amide nucleotide sugars arereadily prepared by this and analogous methods.

The peptide is typically synthesized de novo, or recombinantly expressedin a prokaryotic cell (e.g., bacterial cell, such as E. coli) or in aeukaryotic cell such as a mammalian, yeast, insect, fungal or plantcell. The peptide can be either a full-length protein or a fragment.Moreover, the peptide can be a wild type or mutated peptide. In anexemplary embodiment, the peptide includes a mutation that adds one ormore N- or O-linked glycosylation sites to the peptide sequence.

The method of the invention also provides for modification ofincompletely glycosylated peptides that are produced recombinantly. Manyrecombinantly produced glycoproteins are incompletely glycosylated,exposing carbohydrate residues that may have undesirable properties,e.g., immunogenicity, recognition by the RES. Employing a modified sugarin a method of the invention, the peptide can be simultaneously furtherglycosylated and derivatized with, e.g., a water-soluble polymer,therapeutic agent, or the like. The sugar moiety of the modified sugarcan be the residue that would properly be conjugated to the acceptor ina fully glycosylated peptide, or another sugar moiety with desirableproperties.

Those of skill will appreciate that the invention can be practiced usingsubstantially any peptide or glycopeptide from any source. Exemplarypeptides with which the invention can be practiced are set forth in WO03/031464, and the references set forth therein.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone sugar (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc,glucose, fucose or xylose) to the hydroxy side chain of a hydroxyaminoacid, preferably serine or threonine, although unusual or non-naturalamino acids, e.g., 5-hydroxyproline or 5-hydroxylysine may also be used.

Moreover, in addition to peptides, the methods of the present inventioncan be practiced with other biological structures (e.g., glycolipids,lipids, sphingoids, ceramides, whole cells, and the like, containing aglycosylation site).

Addition of glycosylation sites to a peptide or other structure isconveniently accomplished by altering the amino acid sequence such thatit contains one or more glycosylation sites. The addition may also bemade by the incorporation of one or more species presenting an —OHgroup, preferably serine or threonine residues, within the sequence ofthe peptide (for O-linked glycosylation sites). The addition may be madeby mutation or by full chemical synthesis of the peptide. The peptideamino acid sequence is preferably altered through changes at the DNAlevel, particularly by mutating the DNA encoding the peptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) are preferably madeusing methods known in the art.

In an exemplary embodiment, the glycosylation site is added by shufflingpolynucleotides. Polynucleotides encoding a candidate peptide can bemodulated with DNA shuffling protocols. DNA shuffling is a process ofrecursive recombination and mutation, performed by random fragmentationof a pool of related genes, followed by reassembly of the fragments by apolymerase chain reaction-like process. See, e.g., Stemmer, Proc. Natl.Acad. Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391(1994); and U.S. Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and5,811,238.

Exemplary peptides with which the present invention can be practiced,methods of adding or removing glycosylation sites, and adding orremoving glycosyl structures or substructures are described in detail inWO03/031464 and related U.S. and PCT applications.

The present invention also takes advantage of adding to (or removingfrom) a peptide one or more selected glycosyl residues, after which amodified sugar is conjugated to at least one of the selected glycosylresidues of the peptide. The present embodiment is useful, for example,when it is desired to conjugate the modified sugar to a selectedglycosyl residue that is either not present on a peptide or is notpresent in a desired amount. Thus, prior to coupling a modified sugar toa peptide, the selected glycosyl residue is conjugated to the peptide byenzymatic or chemical coupling. In another embodiment, the glycosylationpattern of a glycopeptide is altered prior to the conjugation of themodified sugar by the removal of a carbohydrate residue from theglycopeptide. See, for example WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically. Anexemplary chemical deglycosylation is brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal. Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

In an exemplary embodiment, the peptide is essentially completelydesialylated with neuraminidase prior to performing glycoconjugation orremodeling steps on the peptide. Following the glycoconjugation orremodeling, the peptide is optionally re-sialylated using asialyltransferase. In an exemplary embodiment, the re-sialylation occursat essentially each (e.g., >80%, preferably greater than 85%, greaterthan 90%, preferably greater than 95% and more preferably greater than96%, 97%, 98% or 99%) terminal saccharyl acceptor in a population ofsialyl acceptors. In a preferred embodiment, the saccharide has asubstantially uniform sialylation pattern (i.e., substantially uniformglycosylation pattern).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N-linked glycosylation, andsites for O-linked glycosylation; (b) terminal glycosyl moieties thatare acceptors for a glycosyltransferase; (c) arginine, asparagine andhistidine; (d) free carboxyl groups; (e) free sulfhydryl groups such asthose of cysteine; (f) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (g) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (h) the amide group ofglutamine. Exemplary methods of use in the present invention aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC CRIT. REV. BIOCHEM., pp. 259-306 (1981).

In one embodiment, the invention provides a method for linking two ormore peptides through a linking group. The linking group is of anyuseful structure and may be selected from straight- and branched-chainstructures. Preferably, each terminus of the linker, which is attachedto a peptide, includes a modified sugar (i.e., a nascent intact glycosyllinking group).

In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a polymeric (e.g., PEGlinker). The construct conforms to the general structure set forth inthe cartoon above. As described herein, the construct of the inventionincludes two intact glycosyl linking groups (i.e., s+t=1). The focus ona PEG linker that includes two glycosyl groups is for purposes ofclarity and should not be interpreted as limiting the identity of linkerarms of use in this embodiment of the invention.

Thus, a PEG moiety is functionalized at a first terminus with a firstglycosyl unit and at a second terminus with a second glycosyl unit. Thefirst and second glycosyl units are preferably substrates for differenttransferases, allowing orthogonal attachment of the first and secondpeptides to the first and second glycosyl units, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby fondling (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².Transferase and/or unreacted peptide is then optionally removed from thereaction mixture. The second peptide and a second transferase for whichthe second glycosyl unit is a substrate are added to the(peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)²; at least one of theglycosyl residues is either directly or indirectly O-linked. Those ofskill in the art will appreciate that the method outlined above is alsoapplicable to forming conjugates between more than two peptides by, forexample, the use of a branched PEG, dendrimer, poly(amino acid),polysaccharide or the like.

In an exemplary embodiment, the peptide that is modified by a method ofthe invention is a glycopeptide that is produced in mammalian cells(e.g., CHO cells) or in a transgenic animal and thus, contains N- and/orO-linked oligosaccharide chains, which are incompletely sialylated. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be PEGylated, PPGylated orotherwise modified with a modified sialic acid.

In Scheme 1, the amino glycoside 1, is treated with the active ester ofa protected amino acid (e.g., glycine) derivative, converting the sugaramine residue into the corresponding protected amino acid amide adduct.The adduct is treated with an aldolase to form α-hydroxy carboxylate 2.Compound 2 is converted to the corresponding CMP derivative by theaction of CMP-SA synthetase, followed by catalytic hydrogenation of theCMP derivative to produce compound 3. The amine introduced via formationof the glycine adduct is utilized as a locus of PEG attachment byreacting compound 3 with an activated PEG or PPG derivative (e.g.,PEG-C(O)NHS, PEG-OC(O)O-p-nitrophenyl), producing species such as 4 or5, respectively.

In an exemplary embodiment, a modified sugar can be attached to anO-glycan binding site on a peptide. The glycosyltransferases which canbe used to produce this conjugate include: for Ser56(-Glc-(Xyl)n-Gal-SA-PEG— a galactosyltransferase and sialyltransferase;for Ser56—Glc-(Xyl)_(n)-Xyl-PEG—a xylosyltransferase; and forSer6O-Fuc-GlcNAc-(Gal)n-(SA)m-PEG—a GlcNAc transferase.

III.A. Conjugation of Modified Sugars to Peptides

The PEG modified sugars are conjugated to a glycosylated ornon-glycosylated peptide using an appropriate enzyme to mediate theconjugation. Preferably, the concentrations of the modified donorsugar(s), enzyme(s) and acceptor peptide(s) are selected such thatglycosylation proceeds until the acceptor is consumed. Theconsiderations discussed below, while set forth in the context of asialyltransferase, are generally applicable to other glycosyltransferasereactions. A list of preferred sialyltransferases for use in theinvention is provided in FIG. 3.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), U.S. Pat. Nos.5,352,670, 5,374,541, 5,545,553, commonly owned U.S. Pat. Nos.6,399,336, and 6,440,703, and commonly owned published PCT applications,WO 03/031464, WO 04/033651, WO 04/099231, which are incorporated hereinby reference.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In a preferred embodiment, each of the first and second enzyme is aglycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycoprotein of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

In another embodiment, the method makes use of one or more exo- orendoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than rupture them. The mutantglycanase typically includes a substitution of an amino acid residue foran active site acidic amino acid residue. For example, when theendoglycanase is endo-H, the substituted active site residues willtypically be Asp at position 130, Glu at position 132 or a combinationthereof. The amino acids are generally replaced with serine, alanine,asparagine, or glutamine.

The mutant enzyme catalyzes the reaction, usually by a synthesis stepthat is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In further embodiments, the GlcNAcresidue on the glycosyl donor molecule is modified. For example, theGlcNAc residue may comprise a 1,2 oxazoline moiety.

In a preferred embodiment, each of the enzymes utilized to produce aconjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above process is carried out can range fromjust above freezing to the temperature at which the most sensitiveenzyme denatures. Preferred temperature ranges are about 0° C. to about55° C., and more preferably about 20° C. to about 37° C. In anotherexemplary embodiment, one or more components of the present method areconducted at an elevated temperature using a thermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few h, withrecoverable amounts usually being obtained within 24 h or less. Those ofskill in the art understand that the rate of reaction is dependent on anumber of variable factors (e.g, enzyme concentration, donorconcentration, acceptor concentration, temperature, solvent volume),which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. As used herein, an industrial scale generallyproduces at least one gram of finished, purified conjugate.

In the discussion that follows, the invention is exemplified by theconjugation of modified sialic acid moieties to a glycosylated peptide.The exemplary modified sialic acid is labeled with PEG. The focus of thefollowing discussion on the use of PEG-modified sialic acid andglycosylated peptides is for clarity of illustration and is not intendedto imply that the invention is limited to the conjugation of these twopartners. One of skill understands that the discussion is generallyapplicable to the additions of modified glycosyl moieties other thansialic acid. Moreover, the discussion is equally applicable to themodification of a glycosyl unit with agents other than PEG includingother PEG moieties, therapeutic moieties, and biomolecules.

An enzymatic approach can be used for the selective introduction ofPEGylated or PPGylated carbohydrates onto a peptide or glycopeptide. Themethod utilizes modified sugars containing PEG, PPG, or a maskedreactive functional group, and is combined with the appropriateglycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

In an exemplary embodiment, an acceptor for a sialyltransferase ispresent on the peptide to be modified either as a naturally occurringstructure or it is placed there recombinantly, enzymatically orchemically. Suitable acceptors, include, for example, galactosylacceptors such as Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc,lacto-N-tetraose, Galβ1,3GlcNAc, Galβ1,3Ara, Galβ1,6GlcNAc, Galβ1,4Glc(lactose), and other acceptors known to those of skill in the art (see,e.g., Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)). Exemplarysialyltransferases are set forth herein.

In one embodiment, an acceptor for the sialyltransferase is present onthe glycopeptide to be modified upon in vivo synthesis of theglycopeptide. Such glycopeptides can be sialylated using the claimedmethods without prior modification of the glycosylation pattern of theglycopeptide. Alternatively, the methods of the invention can be used tosialylate a peptide that does not include a suitable acceptor; one firstmodifies the peptide to include an acceptor by methods known to those ofskill in the art. In an exemplary embodiment, a GalNAc residue is addedby the action of a GalNAc transferase.

In an exemplary embodiment, the galactosyl acceptor is assembled byattaching a galactose residue to an appropriate acceptor linked to thepeptide, e.g., a GlcNAc. The method includes incubating the peptide tobe modified with a reaction mixture that contains a suitable amount of agalactosyltransferase (e.g., Galβ1,3 or Galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

In yet another embodiment, glycopeptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.In another embodiment of this method, the sialic acid moieties of thepeptide are essentially completely removed (e.g., at least 90, at least95 or at least 99%), exposing an acceptor for a modified sialic acid.

In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a PEG moiety attachedthereto. The focus of the discussion is for clarity of illustration.Those of skill will appreciate that the discussion is equally relevantto those embodiments in which the modified sugar bears a therapeuticmoiety, biomolecule or the like.

In an exemplary embodiment of the invention in which a carbohydrateresidue is “trimmed” prior to the addition of the modified sugar highmannose is trimmed back to the first generation biantennary structure. Amodified sugar bearing a PEG moiety is conjugated to one or more of thesugar residues exposed by the “trimming back.” In one example, a PEGmoiety is added via a GlcNAc moiety conjugated to the PEG moiety. Themodified GlcNAc is attached to one or both of the terminal mannoseresidues of the biantennary structure. Alternatively, an unmodifiedGlcNAc can be added to one or both of the termini of the branchedspecies.

In another exemplary embodiment, a PEG moiety is added to one or both ofthe terminal mannose residues of the biantennary structure via amodified sugar having a galactose residue, which is conjugated to aGlcNAc residue added onto the terminal mannose residues. Alternatively,an unmodified Gal can be added to one or both terminal GlcNAc residues.

In yet a further example, a PEG moiety is added onto a Gal residue usinga modified sialic acid such as those discussed above.

In another exemplary embodiment, a high mannose structure is “trimmedback” to the mannose from which the biantennary structure branches. Inone example, a PEG moiety is added via a GlcNAc modified with thepolymer. Alternatively, an unmodified GlcNAc is added to the mannose,followed by a Gal with an attached PEG moiety. In yet anotherembodiment, unmodified GlcNAc and Gal residues are sequentially added tothe mannose, followed by a sialic acid moiety modified with a PEGmoiety.

A high mannose structure can also be trimmed back to the elementarytri-mannosyl core.

In a further exemplary embodiment, high mannose is “trimmed back” to theGlcNAc to which the first mannose is attached. The GlcNAc is conjugatedto a Gal residue bearing a PEG moiety. Alternatively, an unmodified Galis added to the GlcNAc, followed by the addition of a sialic acidmodified with a water-soluble sugar. In yet a further example, theterminal GlcNAc is conjugated with Gal and the GlcNAc is subsequentlyfucosylated with a modified fucose bearing a PEG moiety.

High mannose may also be trimmed back to the first GlcNAc attached tothe Asn of the peptide. In one example, the GlcNAc of theGlcNAc-(Fuc)_(a) residue is conjugated with a GlcNAc bearing a watersoluble polymer. In another example, the GlcNAc of the GlcNAc-(Fuc)_(a)residue is modified with Gal, which bears a water soluble polymer. In astill further embodiment, the GlcNAc is modified with Gal, followed byconjugation to the Gal of a sialic acid modified with a PEG moiety.

Other exemplary embodiments are set forth in commonly owned U.S. Patentapplication Publications: 20040132640; 20040063911; 20040137557; U.S.patent application Ser. Nos. 10/369,979; 10/410,913; 10/360,770;10/410,945 and PCT/US02/32263 each of which is incorporated herein byreference.

The Examples set forth above provide an illustration of the power of themethods set forth herein. Using the methods described herein, it ispossible to “trim back” and build up a carbohydrate residue ofsubstantially any desired structure. The modified sugar can be added tothe termini of the carbohydrate moiety as set forth above, or it can beintermediate between the peptide core and the terminus of thecarbohydrate.

In an exemplary embodiment, an existing sialic acid is removed from aglycopeptide using a sialidase, thereby unmasking all or most of theunderlying galactosyl residues. Alternatively, a peptide or glycopeptideis labeled with galactose residues, or an oligosaccharide residue thatterminates in a galactose unit. Following the exposure of or addition ofthe galactose residues, an appropriate sialyltransferase is used to adda modified sialic acid.

In another exemplary embodiment, an enzyme that transfers sialic acidonto sialic acid is utilized. This method can be practiced withouttreating a sialylated glycan with a sialidase to expose glycan residuesbeneath the sialic acid. An exemplary polymer-modified sialic acid is asialic acid modified with poly(ethylene glycol). Other exemplary enzymesthat add sialic acid and modified sialic acid moieties onto glycans thatinclude a sialic acid residue or exchange an existing sialic acidresidue on a glycan for these species include ST3Gal3, CST-II,ST8Sia-II, ST8Sia-III and ST8Sia-IV.

In yet a further approach, a masked reactive functionality is present onthe sialic acid. The masked reactive group is preferably unaffected bythe conditions used to attach the modified sialic acid to the peptide.After the covalent attachment of the modified sialic acid to thepeptide, the mask is removed and the peptide is conjugated with an agentsuch as PEG. The agent is conjugated to the peptide in a specific mannerby its reaction with the unmasked reactive group on the modified sugarresidue.

Any modified sugar can be used with its appropriate glycosyltransferase,depending on the terminal sugars of the oligosaccharide side chains ofthe glycopeptide. As discussed above, the terminal sugar of theglycopeptide required for introduction of the PEGylated structure can beintroduced naturally during expression or it can be produced postexpression using the appropriate glycosidase(s), glycosyltransferase(s)or mix of glycosidase(s) and glycosyltransferase(s).

In a further exemplary embodiment, UDP-galactose-PEG is reacted withβ1,4-galactosyltransferase, thereby transferring the modified galactoseto the appropriate terminal N-acetylglucosamine structure. The terminalGlcNAc residues on the glycopeptide may be produced during expression,as may occur in such expression systems as mammalian, insect, plant orfungus, but also can be produced by treating the glycopeptide with asialidase and/or glycosidase and/or glycosyltransferase, as required.

In another exemplary embodiment, a GlcNAc transferase, such as GNT1-5,is utilized to transfer PEGylated-GlcNAc to a terminal mannose residueon a glycopeptide. In a still further exemplary embodiment, an the N-and/or O-linked glycan structures are enzymatically removed from aglycopeptide to expose an amino acid or a terminal glycosyl residue thatis subsequently conjugated with the modified sugar. For example, anendoglycanase is used to remove the N-linked structures of aglycopeptide to expose a terminal GlcNAc as a GlcNAc-linked-Asn on theglycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase isused to introduce the PEG-galactose functionality onto the exposedGlcNAc.

In an alternative embodiment, the modified sugar is added directly tothe peptide backbone using a glycosyltransferase known to transfer sugarresidues to the peptide backbone. Exemplary glycosyltransferases usefulin practicing the present invention include, but are not limited to,GalNAc transferases (GalNAc T1-14), GlcNAc transferases,fucosyltransferases, glucosyltransferases, xylosyltransferases,mannosyltransferases and the like. Use of this approach allows thedirect addition of modified sugars onto peptides that lack anycarbohydrates or, alternatively, onto existing glycopeptides. In bothcases, the addition of the modified sugar occurs at specific positionson the peptide backbone as defined by the substrate specificity of theglycosyltransferase and not in a random manner as occurs duringmodification of a protein's peptide backbone using chemical methods. Anarray of agents can be introduced into proteins or glycopeptides thatlack the glycosyltransferase substrate peptide sequence by engineeringthe appropriate amino acid sequence into the polypeptide chain.

In each of the exemplary embodiments set forth above, one or moreadditional chemical or enzymatic modification steps can be utilizedfollowing the conjugation of the modified sugar to the peptide. In anexemplary embodiment, an enzyme (e.g., fucosyltransferase) is used toappend a glycosyl unit (e.g., fucose) onto the terminal modified sugarattached to the peptide. In another example, an enzymatic reaction isutilized to “cap” sites to which the modified sugar failed to conjugate.Alternatively, a chemical reaction is utilized to alter the structure ofthe conjugated modified sugar. For example, the conjugated modifiedsugar is reacted with agents that stabilize or destabilize its linkagewith the peptide component to which the modified sugar is attached. Inanother example, a component of the modified sugar is deprotectedfollowing its conjugation to the peptide. One of skill will appreciatethat there is an array of enzymatic and chemical procedures that areuseful in the methods of the invention at a stage after the modifiedsugar is conjugated to the peptide. Further elaboration of the modifiedsugar-peptide conjugate is within the scope of the invention.

Enzymes and reaction conditions for preparing the conjugates of thepresent invention are discussed in detail in the parent of the instantapplication as well as co-owned published PCT patent applications WO03/031464, WO 04/033651, WO 04/099231.

In a selected embodiment, a peptide expressed in insect cells, isremodeled such that glycans on the remodeled glycopeptide include aGlcNAc-Gal glycosyl residue. The addition of GlcNAc and Gal can occur asseparate reactions or as a single reaction in a single vessel. In thisexample, GlcNAc-transferase I and Gal-transferase I are used. Themodified sialyl moiety is added using ST3Gal-III.

In another embodiment, the addition of GlcNAc, Gal and modified Sia canalso occur in a single reaction vessel, using the enzymes set forthabove. Each of the enzymatic remodeling and glycoPEGylation steps arecarried out individually.

When the peptide is expressed in mammalian cells, different methods areof use. In one embodiment, the peptide is conjugated without need forremodeling prior to conjugation by contacting the peptide with asialyltransferase that transfers the modified sialic acid directly ontoa sialic acid on the peptide forming Sia-Sia-L-R¹, or exchanges a sialicacid on the peptide for the modified sialic acid, forming Sia-L-R¹. Anexemplary enzyme of use in this method is CST-II. Other enzymes that addsialic acid to sialic acid are known to those of skill in the art andexamples of such enzymes are set forth the figures appended hereto.

In yet another method of preparing the conjugates of the invention, thepeptide expressed in a mammalian system is desialylated using asialidase. The exposed Gal residue is sialylated with a modified sialicacid using a sialyltransferase specific for O-linked glycans, providinga peptide with an O-linked modified glycan. The desialylated, modifiedpeptide is optionally partially or fully re-sialylated by using asialyltransferase such as ST3GalIII.

In another aspect, the invention provides a method of making a PEGylatedpeptide conjugate of the invention. The method includes: (a) contactinga peptide comprising a glycosyl group selected from:

with a PEG-sialic acid donor having the formula which is a memberselected from

and an enzyme that transfers PEG-sialic acid from said donor onto amember selected from the GalNAc, Gal and the Sia of said glycosyl group,under conditions appropriate for said transfer. An exemplary modifiedsialic acid donor is CMP-sialic acid modified, through a linker moiety,with a polymer, e.g., a straight chain or branched poly(ethylene glycol)moiety. As discussed herein, the peptide is optionally glycosylated withGalNAc and/or Gal and/or Sia (“Remodeled”) prior to attaching themodified sugar. The remodeling steps can occur in sequence in the samevessel without purification of the glycosylated peptide between steps.Alternatively, following one or more remodeling step, the glycosylatedpeptide can be purified prior to submitting it to the next glycosylationor glycPEGylation step. In an exemplary embodiment, the method furthercomprises expressing the peptide in a host. In an exemplary embodiment,the host is a mammalian cell or an insect cell. In another exemplaryembodiment, the mammalian cell is a member selected from a BHK cell anda CHO cell and the insect cell is a Spodoptera frugiperda cell.

As illustrated in the examples and discussed further below, placement ofan acceptor moiety for the PEG-sugar is accomplished in any desirednumber of steps. For example, in one embodiment, the addition of GalNActo the peptide can be followed by a second step in which the PEG-sugaris conjugated to the GalNAc in the same reaction vessel. Alternatively,these two steps can be carried out in a single vessel approximatelysimultaneously.

In an exemplary embodiment, the PEG-sialic acid donor has the formula:

In another exemplary embodiment, the PEG-sialic acid donor has theformula:

In a further exemplary embodiment, the peptide is expressed in anappropriate expression system prior to being glycopegylated orremodeled. Exemplary expression systems include Sf-9/baculovirus andChinese Hamster Ovary (CHO) cells.

In an exemplary embodiment, the invention provides a method of making apeptide conjugate comprising a glycosyl linker comprising a modifiedsialyl residue having the formula:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from R¹-L- and —C(O)(C₁-C₆)alkyl-R¹; R¹ is a moiety comprisinga member selected from a straight-chain poly(ethylene glycol) residueand branched poly(ethylene glycol) residue; M is a member selected fromH, a metal and a single negative charge; L is a linker which is a memberselected from a bond, substituted or unsubstituted alkyl and substitutedor unsubstituted heteroalkyl, such that when D is OH, G is R′-L-, andwhen G is —C(O)(C₁-C₆)alkyl, D is R′-L-NH—said method comprising: (a) contacting a peptide comprising the glycosylmoiety:

with a PEG-sialic acid donor moiety having the formula:

and an enzyme that transfers said PEG-sialic acid onto the Gal of saidglycosyl moiety, under conditions appropriate for said transfer.

In an exemplary embodiment, L-R¹ has the formula:

wherein a is an integer selected from 0 to 20.

In another exemplary embodiment, R¹ has a structure that is a memberselected from:

wherein e, f, m and n are integers independently selected from 1 to2500; and q is an integer selected from 0 to 20.

Large scale or small scale amounts of peptide conjugate can be producedby the methods described herein. In an exemplary embodiment, the amountof peptide is a member selected from about 0.5 mg to about 100 kg. In anexemplary embodiment, the amount of peptide is a member selected fromabout 0.1 kg to about 1 kg. In an exemplary embodiment, the amount ofpeptide is a member selected from about 0.5 kg to about 10 kg. In anexemplary embodiment, the amount of peptide is a member selected fromabout 0.5 kg to about 3 kg. In an exemplary embodiment, the amount ofpeptide is a member selected from about 0.1 kg to about 5 kg. In anexemplary embodiment, the amount of peptide is a member selected fromabout 0.08 kg to about 0.2 kg. In an exemplary embodiment, the amount ofpeptide is a member selected from about 0.05 kg to about 0.4 kg. In anexemplary embodiment, the amount of peptide is a member selected fromabout 0.1 kg to about 0.7 kg. In an exemplary embodiment, the amount ofpeptide is a member selected from about 0.3 kg to about 1.75 kg. In anexemplary embodiment, the amount of peptide is a member selected fromabout 25 kg to about 65 kg.

The concentration of peptide utilized in the reactions described hereinis a member selected from about 0.5 to about 10 mg peptide/mL reactionmixture. In an exemplary embodiment, the peptide concentration is amember selected from about 0.5 to about 1 mg peptide/mL reactionmixture. In an exemplary embodiment, the peptide concentration is amember selected from about 0.8 to about 3 mg peptide/mL reactionmixture. In an exemplary embodiment, the peptide concentration is amember selected from about 2 to about 6 mg peptide/mL reaction mixture.In an exemplary embodiment, the peptide concentration is a memberselected from about 4 to about 9 mg peptide/mL reaction mixture. In anexemplary embodiment, the peptide concentration is a member selectedfrom about 1.2 to about 7.8 mg peptide/mL reaction mixture. In anexemplary embodiment, the peptide concentration is a member selectedfrom about 6 to about 9.5 mg peptide/mL reaction mixture.

The concentration of CMP-SA-PEG that can be utilized in the reactionsdescribed herein is a member selected from about 0.1 to about 1.0 mM.Factors which may increase or decrease the concentration include thesize of the PEG, time of incubation, temperature, buffer components, aswell as the type, and concentration, of glycosyltransferase used. In anexemplary embodiment, CMP-SA-PEG concentration is a member selected fromabout 0.1 to about 1.0 mM. In an exemplary embodiment, CMP-SA-PEGconcentration is a member selected from about 0.1 to about 0.5 mM. In anexemplary embodiment, CMP-SA-PEG concentration is a member selected fromabout 0.1 to about 0.3 mM. In an exemplary embodiment, CMP-SA-PEGconcentration is a member selected from about 0.2 to about 0.7 mM. In anexemplary embodiment, CMP-SA-PEG concentration is a member selected fromabout 0.3 to about 0.5 mM. In an exemplary embodiment, CMP-SA-PEGconcentration is a member selected from about 0.4 to about 1.0 mM. In anexemplary embodiment, CMP-SA-PEG concentration is a member selected fromabout 0.5 to about 0.7 mM. In an exemplary embodiment, CMP-SA-PEGconcentration is a member selected from about 0.8 to about 0.95 mM. Inan exemplary embodiment, CMP-SA-PEG concentration is a member selectedfrom about 0.55 to about 1.0 mM.

The molar equivalents of CMP-SA-PEG that can be utilized in thereactions described herein are based on the theoretical number ofSA-PEGs that can be added to the protein. The theoretical number ofSA-PEGs is based on the theoretical number of sialation sites on theprotein as well as the MW of the protein when compared to the MW andtherefore moles of CMP-SA-PEG. For a protein, that is about four or fivePEGs based on N-glycans that are primarily bi- and tri-antennary withonly two glycan sites. In an exemplary embodiment, the molar equivalentsof CMP-SA-PEG is an integer selected from 1 to 20. In an exemplaryembodiment, the molar equivalents of CMP-SA-PEG is an integer selectedfrom 1 to 20. In an exemplary embodiment, the molar equivalents ofCMP-SA-PEG is an integer selected from 2 to 6. In an exemplaryembodiment, the molar equivalents of CMP-SA-PEG is an integer selectedfrom 3 to 17. In an exemplary embodiment, the molar equivalents ofCMP-SA-PEG is an integer selected from 4 to 11. In an exemplaryembodiment, the molar equivalents of CMP-SA-PEG is an integer selectedfrom 5 to 20. In an exemplary embodiment, the molar equivalents ofCMP-SA-PEG is an integer selected from 1 to 10. In an exemplaryembodiment, the molar equivalents of CMP-SA-PEG is an integer selectedfrom 12 to 20. In an exemplary embodiment, the molar equivalents ofCMP-SA-PEG is an integer selected from 14 to 17. In an exemplaryembodiment, the molar equivalents of CMP-SA-PEG is an integer selectedfrom 7 to 15. In an exemplary embodiment, the molar equivalents ofCMP-SA-PEG is an integer selected from 8 to 16.

III. B. Simultaneous Desialylation and GlycoPEGylation of a Peptide

The present invention provides a “one-pot” method of glycopegylating apeptide. The one-pot method is distinct from other exemplary processesto make a peptide conjugate, which employ a sequential de-sialylationwith sialidase, subsequent purification of the asialo peptide on ananion exchange column, then glycoPEGylation using CMP-sialic acid-PEGand a glycosyltransferase (such as ST3Gal3), exoglycosidase or anendoglycosidase. The peptide conjugate is then purified via anionexchange followed by size exclusion chromatography to produce thepurified peptide conjugate.

The one-pot method is an improved method to manufacture a peptideconjugate. In this method, the de-sialylation and glycoPEGylationreactions are combined in a one-pot reaction which obviates the firstanion exchange chromatography step used in the previously describedprocess to purify the asialo peptide. This reduction in process stepsproduces several advantages. First, the number of process steps requiredto produce the peptide conjugate is reduced, which also reduces theoperating complexity of the process. Second, the process time for theproduction of the peptide conjugates is reduced e.g., from 4 to 2 days.This reduces the raw material requirements and quality control costsassociated with in-process controls. Third, the invention utilizes lesssialidase, e.g., up to 20-fold less sialidase, e.g., 500 mU/L isrequired to produce the peptide conjugate relative to the process. Thisreduction in the use of sialidase significantly reduces the amount ofcontaminants, such as sialidase, in the reaction mixture.

In an exemplary embodiment, a peptide conjugate is prepared by thefollowing method. In a first step, a peptide is combined with asialidase, a modified sugar of the invention, and an enzyme capable ofcatalyzing the transfer of the glycosyl linking group from the modifiedsugar to the peptide, thus preparing the peptide conjugate. Anysialidase may be used in this method. Exemplary sialidases of use in theinvention can be found in the CAZY database (seehttp://afmb.cnrs-mrs.fr/CAZY/index.html and www.cazy.org/CAZY).Exemplary sialidases can be purchased from any number of sources(QA-Bio, Calbiochem, Manikin, Prozyme, etc.). In an exemplaryembodiment, the sialidase is a member selected from cytoplasmicsialidases, lysosomal sialidases, exo-α sialidases, and endosialidases.In another exemplary embodiment, the sialidase used is produced frombacteria such as Clostridium perfringens or Streptococcus pneumoniae, orfrom a virus such as an adenovirus. In an exemplary embodiment, theenzyme capable of catalyzing the transfer of the glycosyl linking groupfrom the modified sugar to the peptide is a member selected from aglycosyltransferase, such as sialyltransferases and fucosyltransferases,as well as exoglycosidases and endoglycosidases. In an exemplaryembodiment, the enzyme is a glycosyltransferase, which is ST3Gal3. Inanother exemplary embodiment, the enzyme used is produced from bacteriasuch as Escherichia Coli or a fungus such as Aspergillus niger. Inanother exemplary embodiment, the sialidase is added to the peptidebefore the glycosyltransferase for a specified time, allowing thesialidase reaction to proceed before initiating the GlycoPEGylationreaction with addition of the PEG-sialic acid reagent and theglycosyltransferase. Many of these examples are discussed herein.Finally, any modified sugar described herein can be utilized in thisreaction.

In another exemplary embodiment, the method further comprises a‘capping’ step. In this step, additional non-PEGylated sialic acid isadded to the reaction mixture. In an exemplary embodiment, this sialicacid is added to the peptide or peptide conjugate thus preventingfurther addition of PEG-sialic acid. In another exemplary embodiment,this sialic acid impedes the function of the glycosyltransferase in thereaction mixture, effectively stopping the addition of glycosyl linkinggroups to the peptides or peptide conjugates. Most importantly, thesialic acid that is added to the reaction mixture caps theunglycoPEGylated glycans thereby providing a peptide conjugate that hasimproved pharmaceokinetics. In addition, this sialidase can be addeddirectly the glycoPEGylation reaction mixture when the extent ofPEGylation to certain amounts is desired without prior purification.

In an exemplary embodiment, after the capping step, less than about 50%of the sialylation sites on the peptide or peptide conjugate does notcomprise a sialyl moiety. In an exemplary embodiment, after the cappingstep, less than about 40% of the sialylation sites on the peptide orpeptide conjugate does not comprise a sialyl moiety. In an exemplaryembodiment, after the capping step, less than about 30% of thesialylation sites on the peptide or peptide conjugate do not comprise asialyl moiety. In an exemplary embodiment, after the capping step, lessthan about 20% of the sialylation sites on the peptide or peptideconjugate do not comprise a sialyl moiety. In an exemplary embodiment,after the capping step, less than about 10% of the sialylation sites onthe peptide or peptide conjugate do not comprise a sialyl moiety. In anexemplary embodiment, between about 20% and about 5% of the sialylationsites on the peptide or peptide conjugate does not comprise a sialylmoiety. In an exemplary embodiment, between about 25% and about 10% ofthe sialylation sites on the peptide or peptide conjugate does notcomprise a sialyl moiety. In an exemplary embodiment, after the cappingstep, essentially all of the sialylation sites on the peptide or peptideconjugate comprise a sialyl moiety.

III.C. Desialylation and Selective Modification of Peptides

In another exemplary embodiment, the present invention provides a methodfor desialylating a peptide. The method preferably provides a peptidethat is at least about 40%, preferably 45%, preferably about 50%,preferably about 55%, preferably about 60%, preferably about 65%,preferably about 70%, preferably about 75%, preferably about 80%,preferably at least 85%, more preferably at least 90%, still morepreferably, at least 92%, preferably at least 94%, even more preferablyat least 96%, still more preferably at least 98%, and still morepreferably 100% disialylated.

The method includes contacting the peptide with a sialidase, preferablyfor a time period. The preselected time period is sufficient todesialylate the peptide to the degree desired. In a preferredembodiment, the desialylated peptide is separated from the sialidasewhen the desired degree of desialylation is achieved. An exemplarydesialylation reaction and purification cycle is set forth herein.

Those of skill are able to determine an appropriate preselected timeperiod over which to conduct the desialylation reaction. In an exemplaryembodiment, the period is less than 24 hours, preferably less than 8hours, more preferably less than 6 hours, more preferably less than 4hours, still more preferably less than 2 hours and even more preferablyless than 1 hour.

In another exemplary embodiment, in the preparation at the end of thedesialylation reaction, at least 10% of the members of the population ofpeptides has only a single sialic acid attached thereto, preferably atleast 20%, more preferably at least 30%, still more preferably at least40%, even still more preferably at least 50% and more preferably atleast 60%, and still more preferably completely desialylated.

In yet a further exemplary embodiment, in the preparation at the end ofthe desialylation reaction, at least 10% of the members of thepopulation of peptides is fully desialylated, preferably at least 20%,more preferably at least 30%, even more preferably at least 40%, stillmore preferably at least 50% and even still more preferably at least60%.

In still another exemplary embodiment, in the preparation at the end ofthe desialylation reaction, at least 10%, 20%, 30%, 40%, 50% or 60% ofthe members of the peptide population has only a single sialic acid, andat least 10%, 20%, 30%, 40%, 50% or 60% of the peptide is fullydisialylated.

In a preferred embodiment, in the preparation at the end of thedesialylation reaction, at least 50% of the population of peptides isfully disialylated and at least 40% of the members of the peptidepopulation bears only a single sialic acid moiety.

Following desialylation, the peptide is optionally conjugated with asugar or modified sugar. An exemplary modified sugar includes asaccharyl moiety bound to a branched or linear poly(ethylene glycol)moiety. The conjugation is catalyzed by an enzyme that transfers themodified sugar from a modified sugar donor onto an amino acid orglycosyl residue of the peptide. An exemplary modified sugar donor is aCMP-sialic acid that bears a branched or linear poly(ethylene glycol)moiety. An exemplary poly(ethylene glycol) moiety has a molecular weightof at least about 2 KDa, more preferably at least about 5 KDa, morepreferably at least about 10 KDa, preferably at least about 20 KDa, morepreferably at least about 30 KDa, and more preferably at least about 40KDa.

In an exemplary embodiment, the enzyme utilized to transfer the modifiedsugar moiety from the modified sugar donor is a glycosyltransferase,e.g., sialyltransferase. An exemplary sialyltransferase of use in themethods of the invention is ST3Gal3.

An exemplary method of the invention results in a modified peptidebearing at least one, preferably at least two, preferably at least threemodifying groups. In one embodiment, the peptide produced bears a singlemodifying group on the light chain of the peptide. In anotherembodiment, the method provides a modified peptide that bears a singlemodifying group on the heavy chain. In still another embodiment, themethod provides a modified peptide with a single modifying group on thelight chain and a single modifying group on the heavy chain.

In another aspect, the invention provides a method of preparing amodified peptide. The method includes contacting the peptide with amodified sugar donor bearing a modifying group and an enzyme capable oftransferring a modified sugar moiety from the modified sugar donor ontoan amino acid or glycosyl residue of the peptide.

In an exemplary embodiment, the method provides a population of modifiedpeptides in which at least 40%, preferably at least 50%, preferably atleast 60%, more preferably at least 70% and even more preferably atleast 80% of the population members are mono-conjugated on the lightchain of the peptide.

In an exemplary embodiment, the method provides a population of modifiedpeptides in which at least 40%, preferably at least 50%, preferably atleast 60%, more preferably at least 70% and even more preferably atleast 80% of the population members are di-conjugated on the light chainof the peptide.

In an exemplary embodiment of this aspect, the method provides apopulation of modified peptides in which no more than 50%, preferably nomore than 30%, preferably no more than 20%, more preferably no more than10% of the population members are mono-conjugated on the heavy chain ofthe peptide.

In an exemplary embodiment of this aspect, the method provides apopulation of modified peptides in which no more than 50%, preferably nomore than 30%, preferably no more than 20%, more preferably no more than10% of the population members are di-conjugated on the heavy chain ofthe peptide.

The peptide can be subjected to the action of a sialidase prior to thecontacting step, or the peptide can be used without prior desialylation.When the peptide is contacted with a sialidase it can be eitheressentially completely desialylated or only partially desialylated. In apreferred embodiment, the peptide is at least partially desialylatedprior to the contacting step. The peptide may be essentially completelydesialylated (essentially asialo) or only partially desialylated. In apreferred embodiment, the desialylated peptide is one of thedesialylated embodiments described hereinabove.

In yet another aspect, the invention provides a method alteringglycosylation of a peptide. The method includes, (a) contacting thepeptide with a sialidase, thereby removing at least one sialic acidmoiety from the peptide. The peptide can be partially or whollydesialylated as discussed above. In step (b), the product from step (a)with at least one glycosyltransferase and at least one sugar donor. Theat least one sugar donor is a substrate for the at least oneglycosyltransferase. Preferably, step (b) results in transfer of a sugarresidue from the donor onto an amino acid or glycosyl residue of thepeptide. The sugar residue can be a modified or unmodified sugarresidue. The conditions of step (b) are maintained until a desired levelof glycosylation of the peptide is achieved, as discussed above.Following step (b), the sialidase content of the reaction mixture isreduced and preferably, essentially all of the sialidase is removed. Inan exemplary embodiment, the removal of sialidase is effected by achromatography (e.g., ion exchange, e.g., anion exchange) or filtrationstep (e.g., nanofiltration). When the sialidase is removed, theresulting glycosylated peptide (i.e., produced in step (a)) isoptionally contacted with a further at least one sugar donor and atleast one glycosyltransferase, transferring at least one sugar residueonto an amino acid or glycosyl moiety of the peptide. In a preferredembodiment, the sugar residue is sialic acid. In a still furtherpreferred embodiment, the sugar donor is CMP-sialic acid. Theglycosyltransferase is typically a sialyltransferase.

An exemplary sialidase is that of Arthrobacter ureafaciens. Othersialidases of use in the present invention are known in the art. (See,e.g.,http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein&cmd=search&term=sialidase).

Exemplary peptides with which the above method can be practiced arethose set forth in the figures appended hereto, e.g, EPO, Blood Factors(FVII, FVIII, FIX, FXIII, etc.), interferon 1-β, G-CSF, hGH, GLP-1, andBMP-7.

III.D. Additional Aliquots of Reagents Added in the Synthesis of PeptideConjugates

In an exemplary embodiment of the synthesis of the peptide conjugatesdescribed herein, one or more additional aliquots of a reactioncomponent/reagent is added to the reaction mixture after a selectedperiod of time. In another exemplary embodiment, the reactioncomponent/reagent added is a modified sugar nucleotide. Introduction ofa modified sugar nucleotide into the reaction will increase thelikelihood of driving the GlycoPEGylation reaction to completion. In anexemplary embodiment, the nucleotide sugar is a CMP-SA-PEG describedherein. In an exemplary embodiment, the reaction component/reagent addedis a sialidase. In an exemplary embodiment, the reactioncomponent/reagent added is a glycosyltransferase. In an exemplaryembodiment, the reaction component/reagent added is magnesium. In anexemplary embodiment, the additional aliquot added represents about 10%,or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80% or 90% of theoriginal amount in added at the start of the reaction. In an exemplaryembodiment, the reaction component/reagent is added to the reactionabout 3 hours, or 6 hours, or 8 hours, or 10 hours, or 12 hours, or 18hours, or 24 hours, or 30 hours, or 36 hours after its start.

III.E. Selective Production of Light Chain PEGylated Peptide Conjugates

In an exemplary embodiment, the invention provides a method ofincreasing the production of peptide conjugates which are modified onthe light chain over the heavy chain. This method involves theinactivation or sequestering of the heavy chain, thus allowingGlycoPEGylation to preferentially occur on the light chain. The serineprotease activity of the heavy chain of the peptide can be exploited asthe basis for this sequestration. Adding a benzamidine matrix and/orpseudoaffinity resin for serine proteases to a GlycoPEGylation reactionmixture results in sequestration of the heavy chain, whileGlycoPEGylation proceeds on the light chain. The light chain can then bepurified away from the heavy chain by standard techniques known in theart. The heavy chain can be removed from the matrix by the addition ofbenzamidine or removed from the resin by lowering the pH of thesolution. Benzamidine impurities introduced in this step can be removedby diafiltration.

III.E. Purification of Peptide Conjugates

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the productand one or more of the intermediates, e.g., nucleotide sugars, branchedand linear PEG species, modified sugars and modified nucleotide sugars.Standard, well-known techniques for recovery of glycosylated peptidessuch as thin or thick layer chromatography, column chromatography, ionexchange chromatography, or membrane filtration can be used. It ispreferred to use membrane filtration, more preferably utilizing areverse osmotic membrane, or one or more column chromatographictechniques for the recovery as is discussed hereinafter and in theliterature cited herein. For instance, membrane filtration wherein themembranes have molecular weight cutoff of about 3000 to about 10,000 canbe used to remove proteins such as glycosyl transferases. In certaininstances, the molecular weight cutoff differences between the impurityand the product will be utilized in order to ensure productpurification. For example, in order to purify product Peptide-SA-PEG-40KDa from unreacted CMP-SA-PEG-40 KDa, a filter must be chosen that willallow, for example, Peptide-SA-PEG-40 KDa to remain in the retentatewhile allowing CMP-SA-PEG-40 KDa to flow into the filtrate.Nanofiltration or reverse osmosis can then be used to remove saltsand/or purify the product saccharides (see, e.g., WO 98/15581).Nanofilter membranes are a class of reverse osmosis membranes that passmonovalent salts but retain polyvalent salts and uncharged soluteslarger than about 100 to about 2,000 Daltons, depending upon themembrane used. Thus, in a typical application, saccharides prepared bythe methods of the present invention will be retained in the membraneand contaminating salts will pass through.

If the peptide is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, is removed.Following glycoPEGylation, the PEGylated peptide is purified byart-recognized methods, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the polypeptide variant from other impurities by one or moresteps selected from immunoaffinity chromatography, ion-exchange columnfractionation (e.g., on diethylaminoethyl (DEAE) or matrices containingcarboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose,CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose,WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, PhenylToyopearl, or protein A Sepharose, SDS-PAGE chromatography, silicachromatography, chromatofocusing, reverse phase HPLC (e.g., silica gelwith appended aliphatic groups), gel filtration using, e.g., Sephadexmolecular sieve or size-exclusion chromatography, chromatography oncolumns that selectively bind the polypeptide, and ethanol or ammoniumsulfate precipitation. Purification can be used to separate one chain ofthe peptide conjugate from the other, as further described later in thissection.

Modified glycopeptides produced in culture are usually isolated byinitial extraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps. Additionally, the modified glycoprotein may bepurified by affinity chromatography. Finally, HPLC may be employed forfinal purification steps.

A protease inhibitor may be included in any of the foregoing steps toinhibit proteolysis and antibiotics or preservatives may be included toprevent the growth of adventitious contaminants. The protease inhibitorsused in the foregoing steps may be low molecular weight inhibitors,including antipain, alpha-1-antitrypsin, anti-thrombin, leupeptin,amastatin, chymostatin, banzamidin, as well as other serine proteaseinhibitors (i.e. serpins). Generally, serine protease inhibitors shouldbe used in concentrations ranging from 0.5-100 μM, although chymostatinin cell culture may be used in concentrations upward of 200 μM. Otherserine protease inhibitors will include inhibitors specific to thechymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, or thesignal peptidase clans of serine proteases. Besides serine proteases,other types of protease inhibitors may also be used, including cysteineprotease inhibitors (1-10 μM) and aspartic protease inhibitors (1-5 μM),as well as non-specific protease inhibitors such as pepstatin (0.1-5μM). Protease inhibitors used in this invention may also include naturalprotease inhibitors, such as the hirustasin inhibitor isolated fromleech. In some embodiments, protease inhibitors will comprise syntheticpeptides or antibodies that are able to bind with specificity to theprotease catalytic site to stabilize the peptide without interferingwith a glycoPEGylation reaction.

Within another embodiment, supernatants from systems which produce themodified glycopeptide of the invention are first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Other methods of use in purification include size exclusionchromatography (SEC), hydroxyapatite chromatography, hydrophobicinteraction chromatography and chromatography on Blue Sepharose. Theseand other useful methods are illustrated in co-assigned U.S. ProvisionalPatent No. (Attorney Docket No. 40853-01-5168-P1, filed May 6, 2005).

One or more RP-HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide conjugate composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous or essentially homogeneousmodified glycoprotein.

The modified glycopeptide of the invention resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et al., J. Chromatog. 296: 171 (1984). This reference describestwo sequential, RP-HPLC steps for purification of recombinant human IL-2on a preparative HPLC column. Alternatively, techniques such as affinitychromatography may be utilized to purify the modified glycoprotein.

In an exemplary embodiment, the purification is accomplished by themethods set forth in commonly owned, co-assigned U.S. Provisional PatentNo. 60/665,588, filed Mar. 24, 2005.

According to the present invention, pegylated peptides are producedeither via sequential de-sialylation or simultaneous sialylation can bepurified or resolved by using magnesium chloride gradient.

In an exemplary embodiment, the peptide conjugates can be separated intoa light chain and a heavy chain, and one chain can be purified away fromthe other. In another exemplary embodiment, a product is obtained inwhich at least 80% of the peptide conjugate in the product is the lightchain portion of the peptide conjugate. In another exemplary embodiment,a product is obtained in which at least 90% of the peptide conjugate inthe product is the light chain portion of the peptide conjugate. Inanother exemplary embodiment, a product is obtained in which at least95% of the peptide conjugate in the product is the light chain portionof the peptide conjugate. In another exemplary embodiment, a product isobtained in which essentially all of the peptide conjugate in theproduct is the light chain portion of the peptide conjugate. Thisproduct is possible for any compound of the invention.

In another exemplary embodiment, a product is obtained in which at least80% of the peptide conjugate in the product is the heavy chain portionof the peptide conjugate. In another exemplary embodiment, a product isobtained in which at least 90% of the peptide conjugate in the productis the heavy chain portion of the peptide conjugate. In anotherexemplary embodiment, a product is obtained in which at least 95% of thepeptide conjugate in the product is the heavy chain portion of thepeptide conjugate. In another exemplary embodiment, a product isobtained in which essentially all of the peptide conjugate in theproduct is the heavy chain portion of the peptide conjugate. Thisproduct is possible for any compound of the invention.

III.F. Properties of Peptide Conjugates

In an exemplary embodiment, the peptide conjugates of the inventionpossess essentially the same biochemical properties as a native peptide.In an exemplary embodiment, the peptide conjugates of the inventionpossess reduced, or enhanced biochemical properties over a nativepeptide depending on the site of PEGylation, the size of the PEG addedand the number of PEGs added.

IV. Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical composition.The pharmaceutical composition includes a pharmaceutically acceptablediluent and a covalent conjugate between a non-naturally-occurring, PEGmoiety, therapeutic moiety or biomolecule and a glycosylated ornon-glycosylated peptide. The polymer, therapeutic moiety or biomoleculeis conjugated to the peptide via an intact glycosyl linking groupinterposed between and covalently linked to both the peptide and thepolymer, therapeutic moiety or biomolecule.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

In an exemplary embodiment, the pharmaceutical formulation comprises apeptide conjugate and a pharmaceutically acceptable diluent which is amember selected from sodium chloride, calcium chloride dihydrate,glycylglycine, polysorbate 80, and mannitol. In another exemplaryembodiment, the pharmaceutically acceptable diluent is sodium chlorideand glycylglycine. In another exemplary embodiment, the pharmaceuticallyacceptable diluent is calcium chloride dihydrate and polysorbate 80. Inanother exemplary embodiment, the pharmaceutically acceptable diluent ismannitol.

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administered parenterally,e.g., intravenously. Thus, the invention provides compositions forparenteral administration that include the compound dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier, e.g.,water, buffered water, saline, PBS and the like. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the glycopeptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface.

The compounds prepared by the methods of the invention may also find useas diagnostic reagents. For example, labeled compounds can be used tolocate areas of inflammation or tumor metastasis in a patient suspectedof having an inflammation. For this use, the compounds can be labeledwith ¹²⁵I, ¹⁴C, or tritium.

The active ingredient used in the pharmaceutical compositions of thepresent invention is peptide conjugates having the biological propertiesof stimulating blood clot production. Preferably, the peptide conjugateare administered parenterally. Effective dosages are expected to varyconsiderably depending on the condition being treated and the route ofadministration but are expected to be in the range of about 0.1 (˜7U) to100 (˜7000U) μg/kg body weight of the active material. Preferable dosesfor treatment of anemic conditions are about 50 to about 300 Units/kgthree times a week. Because the present invention provides a compositionof matter comprising a peptide with an enhanced in vivo residence time,the stated dosages are optionally lowered when a composition of theinvention is administered.

Preparative methods for species of use in preparing the compositions ofthe invention are generally set forth in various patent publications,e.g., US 20040137557; WO 04/083258; and WO 04/033651. The followingexamples are provided to illustrate the conjugates, and methods and ofthe present invention, but not to limit the claimed invention.

EXAMPLES Example 1 Purification of PEGylated Factor VIIa

GlycoPEGylated samples of Factor VIIa were purified with a modifiedanion-exchange method. Samples were handled at 5° C. Immediately beforeloading the column, 1 g Chelex 100 (BioRad) per 10 mL Factor VIIasolution was added to the remodeled sample. After stirring for 10 min,the suspension was filtered on a cellulose acetate membrane (0.2 μm)with a vacuum system. The retained chelator resin on the filter waswashed once with 1-2 mL water per 10 mL bulk. The conductivity of thefiltrate was adjusted to 10 mS/cm at 5° C., and adjusted to pH 8.6, ifnecessary.

Anion exchange was performed at 8-10° C. A column containing Q SepharoseFF was prepared before loading by washing with 1 M NaOH (10 columnvolumes), water (5 column volumes), 2 M NaCl, 50 mM HOAc, pH 3 (10column volumes), and equilibrating with 175 mM NaCl, 10 mMglycylglycine, pH 8.6 (10 column volumes). For each PEGylation reaction,15-20 mg Factor VIIa was loaded on to an XK16 column (AmershamBiosciences) with 10 mL Q Sepharose FF (no more than 2 mg protein per mLresin) at a flow rate of 100 cm/h. For the 2 KDa linear PEG, 20 mgFactor VIIa was loaded on to an XK26 column (Amersham Biosciences) with40 mL Q Sepharose FF (0.5 mg protein per mg resin) at a flow rate of 100cm/h.

After loading, the column was washed with 175 mM NaCl, 10 mMglycylglycine, pH 8.6 10 column volumes) and 50 mM NaCl, 10 mMglycylglycine, pH 8.6 (2 column volumes). Elution was performed with astep gradient of 15 mM CaCl₂ by using 50 mM NaCl, 10 mM glycylglycine,15 mM CaCl₂, pH 8.6 (5 column volumes). The column was then washed with1 M NaCl, 10 mM glycylglycine, pH 8.6 (5 column volumes). The effluentwas monitored by absorbance at 280 nm. Fractions (5 mL) were collectedduring the flow-through and the two washes; 2.5 mL fractions werecollected during the CaCl₂ and 1M salt elutions. Fractions containingFactor VIIa were analyzed by non-reducing SDS-PAGE (Tris-glycine gelsand/or NUPAGE gels) and a Colloidal Blue Staining Kit. The appropriatefractions with Factor VIIa were pooled, and the pH was adjusted to 7.2with 4 M HCl.

Factor VIIa-SA-PEG-10 KDa was purified as described above, except forthe following changes. EDTA (10 mM) was added to to the PEGylated FactorVIIa solution, the pH was adjusted to pH 6, and the conductivity wasadjusted to 5 mS/cm, at 5° C. About 20 mg of Factor VIIa-SA-PEG-10 KDawas loaded on to an XK16 column (Amersham Biosciences) with 10 mL Poros50 Micron HQ resin (no more than 2 mg protein per mL, resin) at a flowrate of 100 cm/h. After loading, the column was washed with 175 mM NaCl,10 mM histidine pH 6 (10 column volumes) and 50 mM NaCl, 10 mMhistidine, pH 6 (2 column volumes). Elution was performed with a stepgradient of 20 mM CaCl₂ in 50 mM NaCl, 10 mM histidine, pH 6 (5 columnvolumes). The column was then washed with 1 M NaCl, 10 mM histidine, pH6 (5 column volumes).

The anion-exchange eluate containing Factor VIIa-SA-PEG-10 KDa (25 mL)was concentrated to 5-7 mL by using an Amicon Ultra-15 10K centrifugalfilter device, according to the manufacturer's directions (Millipore).Following concentration, size exclusion chromatography was performed.The sample (5-7 mL) was loaded onto a column containing Superdex 200(HiLoad 16/60, prep grade; Amersham Biosciences) equilibrated in 50 mMNaCl, 10 mM glycylglycine, 15 mM CaCl₂, pH 7.2 for most of the PEGylatedvariants. Factor VIIa-SA-PEG-10 KDa was separated from the unmodified,asialo-Factor VIIa at a flow rate of 1 mL/min, and the absorbance wasmonitored at 280 nm. Fractions (1 mL) containing Factor VIIa werecollected and analyzed by non-reducing SDS-PAGE (Tris-glycine gelsand/or NuPAGE gels) and a Colloidal Blue Staining Kit. Fractionscontaining the targeted PEGylated isoform and devoid of the unmodified,asialo-Factor VIIa were pooled and concentrated to 1 mg/mL using anAmicon Ultra-15 10K centrifugal filter device. Protein concentration wasdetermined from absorbance readings at 280 nm using an extinctioncoefficient of 1.37 (mg/mL)⁻¹ cm⁻¹.

Example 2 Determination of PEGylated Isoforms by Reversed phase HPLCanalysis

PEGylated Factor VIIa was analyzed by HPLC on a reversed-phase column(Zorbax 300SB-C3, 5 μm particle size, 2.1×150 mm). The eluants were A)0.1 TFA in water and B) 0.09% TFA in acetonitrile. Detection was at 214nm. The gradient, flow rate, and column temperature depended on the PEGlength (40 KDa, 20 KDa, and 10 KDa PEG: 35-65% B in 30 min, 0.5 mL/min,45° C.; 10 KDa PEG: 35-60% B in 30 min, 0.5 mL/min, 45° C.; 5 KDa:40-50% B in 40 min, 0.5 mL/min, 45° C.; 2 KDa: 38-43% B in 67 min, 0.6mL/min, 55° C.). The identity of each peak was assigned based on two ormore of four different pieces of evidence: the known retention time ofnative Factor VIIa, the SDS-PAGE migration of the isolated peak, theMALDI-TOF mass spectrum of the isolated peak, and the orderlyprogression of the retention time of each peak with increasing number ofattached PEG.

Example 3 Determination of Site of PEG Attachment by Reversed-Phase HPLC

Factor VIIa and PEGylated Factor VIIa variants were reduced by mixingsample (10 μL at a concentration of 1 mg/mL) with reducing buffer (40μL, 50 mM NaCl, 10 mM glycylglycine, 15 mM EDTA, 8 M urea, 20 mM DTT, pH8.6) for 15 min at room temperature. Water (50 μL) was added and thesample cooled to 4° C. until injected on the HPLC (<12 hrs). The HPLCcolumn, eluants, and detection were as described above for non-reducedsamples. The flow rate was 0.5 mL/min and the gradient was 30-55% B in90 min, followed by a brief wash cycle up to 90% B. The identity of eachpeak was assigned as described in Example 2.

Example 4 Factor VIIa Clotting Assay

PEGylated samples and standards were tested in duplicate, and werediluted in 100 mM NaCl, 5 mM CaCl₂. 0.1% BSA (wt/vol), 50 mM Tris, pH7.4. The standard and samples were assayed over a range from 0.1 to 10ng/mL. Equal volumes of diluted standards and samples were mixed withFactor VIIa deficient plasma (Diagnostica Stago), and stored on ice forno greater than 4 hours before they were assayed.

Clotting times were measured with a STart4 coagulometer (DiagnosticaStago). The coagulometer measured the time elapsed until an in vitroclot was formed, as indicated by the stopping of the gentleback-and-forth movement of a magnetic ball in a sample cuvette.

Into each cuvette, one magnetic ball was deposited, plus 100 μL FactorVIIa sample/deficient plasma and 100 μL of a diluted rat brain cephalinsolution (stored on ice for no greater than 4 hours). Each reagent wasadded with 5 seconds between each well, and the final mixture wasincubated for 300 seconds at 37° C. Diluted rat brain cephalin (RBC)solution was made from 2 mL RBC stock solution (1 vial RBC stock, fromHaemachem, plus 10 mL 150 mM NaCl) and 4 mL 100 mM NaCl, 5 mM CaCl₂,0.1% BSA (wt/vol), 50 mM Tris, pH 7.4.

At 300 seconds, the assay was started by the addition of 100 μl, of apre-heated (37° C.) solution of soluble tissue factor (2 μg/mL; aminoacids 1-209) in 100 mM NaCl. 12.5 mM CaCl₂, 0.1% BSA (wt/vol), 50 mMTris, pH 7.4. Again, this next solution was added with a 5 secondinterval between samples.

The clotting times from the diluted standards were used to generate astandard curve (log clot time versus log Factor VIIa concentration). Theresulting linear regression from the curve was used to determine therelative clotting activities of PEGylated variants. PEGylated FactorVIIa variants were compared against an aliquotted stock of Factor VIIa.

Example 5 Factor VIIa-SA-PEG-10 KDa: One Pot Method

Factor VIIa (5 mg diluted in the product formulation buffer to a finalconcentration of 1 mg/mL), CMP-SA-PEG-10 KDa (10 mM, 60 μL) and A. nigerenzyme ST3Gal3 (33 U/L) and 10 mM histidine, 50 mM NaCl, 20 mM CaCl₂were combined in a reaction vessel along with either 10 U/L, 1 U/L, 0.5U/L or 0.1 U/L of sialidase (CalBiochem). The ingredients were mixed andincubated at 32° C. Reaction progress was measured by analyzing aliquotsat 30 minute intervals for the first four hours. An aliquot was thenremoved at the 20 hour timepoint and subjected to SDS-PAGE. Extent ofPEGylation was determined by removing 1 mL at 1.5, 2.5 and 3.5 hourtimepoint and purifying the sample on a Poros 50HQ column.

For the reaction conditions containing 10 U/L of sialidase, noappreciable amount of Factor VIIa-SA-PEG product was formed. For thereaction conditions containing 1 U/L of sialidase, about 17.6% of theFactor VIIa in the reaction mixture was either mono or diPEGylated after1.5 hours. This increased to 29% after 2.5 hours, and 40.3% after 3.5hours. For the reaction conditions containing 0.5 U/L of sialidase,about 44.5% of the Factor VIIa in the reaction mixture was either monoor diPEGylated after 3 hours, and 0.8% was triPEGylated or greater.After 20 hours, 69.4% was either mono or diPEGylated, and 18.3% wastriPEGylated or greater.

For the reaction conditions containing 0.1 U/L of sialidase, about 29.6%of the Factor VIIa in the reaction mixture was either mono ordiPEGylated after 3 hours. After 20 hours, 71.3% was either mono ordiPEGylated, and 15.1% was triPEGylated or greater.

Results are shown in FIG. 11 and FIG. 12.

Example 6 Preparation of Cysteine-PEG₂ (2)

a. Synthesis of Compound 1

Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was added to asolution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 L)under argon. The mixture was stirred at room temperature for 30 min, andthen mPEG-O-tosylate of molecular mass 20 kilodalton (Ts; 1.0 g, 0.05mmol) was added in several portions over 2 hours. The mixture wasstirred at room temperature for 5 days, and concentrated by rotaryevaporation. The residue was diluted with water (30 mL), and stirred atroom temperature for 2 hours to destroy any excess 20 kilodaltonmPEG-O-tosylate. The solution was then neutralized with acetic acid, thepH adjusted to pH 5.0 and loaded onto a reverse phase chromatography(C-18 silica) column. The column was eluted with a gradient ofmethanol/water (the product elutes at about 70% methanol), productelution monitored by evaporative light scattering, and the appropriatefractions collected and diluted with water (500 mL). This solution waschromatographed (ion exchange, XK 50 Q, BIG Beads, 300 mL, hydroxideform; gradient of water to water/acetic acid-0.75N) and the pH of theappropriate fractions lowered to 6.0 with acetic acid. This solution wasthen captured on a reversed phase column (C-18 silica) and eluted with agradient of methanol/water as described above. The product fractionswere pooled, concentrated, redissolved in water and freeze-dried toafford 453 mg (44%) of a white solid (1).

Structural data for the compound were as follows: ¹H-NMR (500 MHz; D₂O)δ 2.83 (t, 2H, O—C—CHhd 2—S), 3.05 (q, 1H, S—CHH—CHN), 3.18 (q, 1H, (q,1H, S—CHH—CHN), 3.38 (s, 3H, CH ₃O), 3.7 (t, OCH₂ CH₂ O), 3.95 (q, 1H,CHN). The purity of the product was confirmed by SDS PAGE.

b. Synthesis of Cysteine-PEG₂ (2)

Triethylamine (˜0.5 mL) was added dropwise to a solution of compound 1(440 mg, 22 μmol) dissolved in anhydrous CH₂Cl₂ (30 mL) until thesolution was basic. A solution of 20 kilodalton mPEG-O-p-nitrophenylcarbonate (660 mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 !mopin CH₂Cl₂ (20 mL) was added in several portions over 1 hour at roomtemperature. The reaction mixture was stirred at room temperature for 24hours. The solvent was then removed by rotary evaporation, the residuewas dissolved in water (100 mL), and the pH adjusted to 9.5 with 1.0 NNaOH. The basic solution was stirred at room temperature for 2 hours andwas then neutralized with acetic acid to a pH 7.0. The solution was thenloaded onto a reversed phase chromatography (C-18 silica) column. Thecolumn was eluted with a gradient of methanol/water (the product elutesat about 70% methanol), product elution monitored by evaporative lightscattering, and the appropriate fractions collected and diluted withwater (500 mL). This solution was chromatographed (ion exchange, XK 50Q, BIG Beads, 300 mL, hydroxide faun; gradient of water to water/aceticacid-0.75N) and the pH of the appropriate fractions lowered to 6.0 withacetic acid. This solution was then captured on a reversed phase column(C-18 silica) and eluted with a gradient of methanol/water as describedabove. The product fractions were pooled, concentrated, redissolved inwater and freeze-dried to afford 575 mg (70%) of a white solid (2).

Structural data for the compound were as follows: ¹H-NMR (500 MHz; D₂O)δ 2.83 (t, 2H, O—C—CHhd 2—S), 2.95 (t, 2H, O—C-CHhd 2—S), 3.12 (q, 1H,S—CHH—CHN), 3.39 (s, 3H CH ₃O), 3.71 (t, OCH₂CH₂O). The purity of theproduct was confirmed by SDS PAGE.

Example 7 Factor VIIa-SA-PEG-40 KDa

GlycoPEGylation of Factor VIIa (One Pot with Capping). GlycoPEGylationof Factor VIIa was accomplished in a one-pot reaction where desialationand PEGylation occur simultaneously, followed by capping with sialicacid. The reaction was performed in a jacketed glass vessel controlledat 32° C. by a recirculating waterbath. First, the concentrated 0.2μm-filtered Factor VIIa was introduced into the vessel and heated to 32°C. by mixing with a stir bar for 20 minutes. A solution of sialidase wasmade from dry powder in 10 mM histidine/50 mM NaC1/20 mM CaCl₂, pH 6.0at a concentration of 4,000 U/L. Once the Factor VIIa reached 32° C.,the sialidase was added to the Factor VIIa, and the reaction was mixedfor approximately 5 minutes to ensure a uniform solution after timewhich the mixing was stopped. The desialation was allowed to proceed for1.0 h at 32° C. During the desialation reaction, the CMP-SA-PEG-40 KDawas dissolved into 10 mM histidine/50 mM NaC1/20 mM CaCl₂, pH 6.0buffer, and the concentration of was determined by UV absorbance at 271nm. After the CMP-SA-PEG-40 KDa was dissolved, the CMP-SA-PEG-40 KDa wasadded to the reaction, as well as the ST3Gal3, and the reaction wasmixed for approximately 15 minutes with a stir bar to ensure a uniformsolution. An additional volume of 85 mL of buffer was added to make thereaction 1.0 L. The reaction was allowed to proceed without stirring for24 hours before CMP-SA was added to a concentration of 4.3 mM to quenchthe reaction and cap the remaining terminal galactose residues withsialic acid. The quenching was allowed to proceed with mixing for 30minutes at 32° C. The total volume of the reaction was 1.0 L beforequenching. Timepoint samples (1 mL) were taken at 0, 4.5, 7.5, and 24 h,quenched with CMP-SA, and analyzed by RP-HPLC and SDS-PAGE.

Purification of Factor VIIa-SA-PEG-40 KDa. After capping, the solutionwas diluted with 2.0 L of 10 mM histidine, pH 6.0 that had been storedovernight at 4° C. and the sample was filtered through a 0.2 μm Millipak60 filter. The resulting load volume was 3.1 L. The AEX2 chromatographywas performed at 20-25° C. (ambient room temperature) on an Akta Pilotsystem. After loading, a 10 column volumes wash with equilibrationbuffer was performed, and the product was eluted from the column using a10 column volume gradient of MgCl₂ which resulted in resolution ofPEGylated-Factor VIIa species from unPEGylated Factor VIIa. The loadingfor this column was intentionally kept low, targeting <2 mg FactorVIIa/mL resin. SDS-PAGE gels were run in addition to RP-HPLC analysis ofselected fractions and pools of fractions in order to make the pool ofbulk product. Pooled fractions were pH adjusted to 6.0 with 1M NaOH andstored in the cold room at 2-8° C. overnight.

Final Concentration/Diafiltration, aseptic filtration and aliquoting.The pooled fractions were filtered through a Millipak 20 0.2 μm filterand stored overnight at 2-8° C. To perform theconcentration/diafiltration, a Millipore 0.1m² 30 KDa regeneratedcellulose membrane was used in a system fitted with a peristaltic pumpand silicone tubing. The system was assembled and flushed with water,then sanitized with 0.1 M NaOH for at least 1 hour, and then stored in0.1M NaOH until equilibration with 10 mM histidine/5 mM CaCl₂/100 mMNaCl pH 6.0 diafiltration buffer immediately before use. The product wasconcentrated to approximately 400 mL and then diafiltered at constantvolume with approximately 5 diavolumes of buffer. The product was thenconcentrated to approximately 300 mL and recovered after a low pressurerecirculation for 5 minutes, and the membranes were rinsed with 200 mLof diafiltration buffer by a recirculation for 5 minutes. The wash wasrecovered with product, and another 50 mL of buffer was recirculated foranother 5 minutes for a final wash. The resulting bulk was approximately510 mL, and that was filtered through a 1L vacuum filter fitted with a0.2 μm PES membrane (Millipore). The aseptically-filtered bulk was thenaliquoted into 25 mL aliquots in 50 mL sterile falcon tubes and frozenat −80° C.

Analysis of the PEGylation Reaction by HPLC (Example 7)

Purification Conjugation Reaction Time After 0 hrs 4.5 hrs 7.5 hrs 24hrs Chromatography % Unpegylated 94.7 76.1 66.6 51.0 0.6 % Monopegylated0.9 17.9 26.1 39.1 85.6 % Dipegylated 0.1 0.9 1.9 5.1 5.1 % Tripegylated0.0 0.0 0.0 0.2 0.2After 24 hours, the bulk product PEG-state distribution was: 0.7%unpegylated, 85.3% mono-pegylated, 11.5% di-pegylated, and 0.3%tri-pegylated. Column chromatography is the main step in the processthat generates the product distribution, largely through removingunpegylated material from mono- and di-pegylated species.

Example 8 Factor VIIa-SA-PEG-10 KDa

The following example describes a procedure for determining the numberof modified sugar attachments to light and heavy chains of FactorVIIa-SA-PEG-10 KDa by reverse phase HPLC.

Factor VIIa-SA-PEG-10 KDa was subjected to reducing conditions in orderto separate the heavy chain from the light chain. After separation, theheavy and light chains were subjected to separate reverse phase HPLCexperiments. Peaks were assigned based on their position relative to thenon-modified Factor VIIa peaks in the chromatograms of the native FactorVIIa control.

The following table describes the HPLC solvent gradient parameters forthe light chain. The column temperature was 39° C.

HPLC Light Chain Solvent Gradient Parameters

Time, min Solvent B, % Flow rate, mL/min Comment 0 30 0.5 Initialcondition 60 47 0.5 Gradient elution 60.2 90 0.5 Start wash 70 90 0.5Wash

The chromatograms of light chain Factor VIIa-SA-PEG-10 KDa (top) andnative light chain Factor VIIa (bottom) are provided in FIG. 14A.

The following table describes the HPLC solvent gradient parameters forthe heavy chain. The column temperature was 52° C.

HPLC Heavy Chain Solvent Gradient Parameters

Time, min Solvent B, % Flow rate, ml/min Comment 0 42.5 0.5 Initialcondition 36 52.5 0.5 Gradient elution 36.1 90 0.5 Start wash 41 90 0.5wash

The chromatograms of heavy chain Factor VIIa-SA-PEG-10 KDa (top) andnative heavy chain Factor VIIa (bottom) are provided in FIG. 14B.

Example 9 Factor VIIa-SA-PEG-40 KDa

The following example describes a procedure for determining the numberof modified sugar attachments to light and heavy chains of FactorVIIa-SA-PEG-40 KDa by reverse phase HPLC.

Factor VIIa-SA-PEG-40 KDa was subjected to reducing conditions in orderto separate the heavy chain from the light chain. After separation, theheavy and light chains were subjected to separate reverse phase HPLCexperiments. Peaks were assigned based on their position relative to thenon-modified sugar peaks in the chromatograms of the native Factor VIIacontrol.

The following table describes the HPLC solvent gradient parameters forthe light chain. The column temperature was 25° C.

HPLC Light Chain Solvent Gradient Parameters

Time (min) Eluent B (%) Comment 0 30 Initial conditions 60 47 Gradientelution 60.5 90 Begin wash 65.5 90 End wash 66 42.5 Begin heavy chainmethod equilibration 70 42.5 End of Run

The chromatograms of light chain Factor VIIa-SA-PEG-40 KDa (bottom) andnative light chain Factor VIIa (top) are provided in FIG. 15A.

The following table describes the HPLC solvent gradient parameters forthe heavy chain. The column temperature was 40° C.

HPLC Heavy Chain Solvent Gradient Parameters

Time (min) Eluent B (%) Comment 0 42.5 Initial conditions 36 52.5Gradient elution 36.5 90 Begin wash 41.5 90 End wash 42 30 Begin lightchain method equilibration 47 30 End Run

The chromatograms of heavy chain Factor VIIa-SA-PEG-40 KDa (bottom) andnative heavy chain Factor VIIa (top) are provided in FIG. 15B.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-35. (canceled)
 36. A method of synthesizing a peptide conjugate comprising combining: (a) sialidase; (b) an enzyme capable of catalyzing the transfer of a glycosyl linking group; (c) a modified sugar; and (d) a peptide, under conditions appropriate to form a peptide conjugate, thereby synthesizing the peptide conjugate.
 37. The method of claim 36, wherein a covalent bond is formed between the modified sugar and the peptide.
 38. The method of claim 36, wherein the method comprises (i) a desialylation step in which the sialidase removes a sialic acid residue from the peptide, thereby forming a desialyated peptide, and (ii) a conjugation step in which the enzyme capable of catalyzing the transfer of a glycosyl linking group transfers a modified sugar moiety from the modified sugar to an amino acid or glycosyl residue of the peptide, which desialylation and conjugation steps are performed in the same vessel.
 39. The method of claim 38, wherein the desialyated peptide is not purified prior to the conjugation step.
 40. The method of claim 39, wherein the enzyme capable of catalyzing the transfer of a glycosyl linking group is a glycosyltransferase.
 41. The method of claim 36, wherein the peptide is in contact with components (a)-(c) simultaneously.
 42. The method of claim 37, wherein the peptide is in contact with components (a)-(c) simultaneously.
 43. The method of claim 38, wherein the peptide is in contact with components (a)-(c) simultaneously.
 44. The method of claim 39, wherein the peptide is in contact with components (a)-(c) simultaneously.
 45. The method of claim 40, wherein the peptide is in contact with components (a)-(c) simultaneously.
 46. The method of claim 36, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.
 47. The method of claim 37, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.
 48. The method of claim 38, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.
 49. The method of claim 39, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.
 50. The method of claim 40, wherein the sialidase is in contact with the modified sugar and the peptide for 30 minutes to two hours prior to being in contact with the enzyme capable of catalyzing the transfer of a glycosyl linking group.
 51. The method of claim 36, wherein the enzyme capable of catalyzing the transfer of a glycosyl linking group is selected from the group consisting of glycosyltransferase, exoglycosidase, and endogylcosidase.
 52. The method of claim 51, wherein the combining of components (a)-(d) is for a time less than ten hours.
 53. The method of claim 36, wherein the modified sugar comprises a sugar moiety, and the sugar moiety is a nucleotide sugar.
 54. The method of claim 53, wherein the nucleotide sugar comprises poly(ethylene glycol).
 55. The method of claim 54, wherein at least 50% of the peptide conjugate includes at least two poly(ethylene glycol) moieties.
 56. The method of claim 54, wherein at least 50% of the peptide conjugate includes at most one poly(ethylene glycol) moiety.
 57. The method of claim 53, wherein the nucleotide sugar is selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, and CMP-NeuAc.
 58. The method of claim 57, wherein the nucleotide sugar comprises poly(ethylene glycol).
 59. The method of claim 58, wherein at least 50% of the peptide conjugate includes at least two poly(ethylene glycol) moieties.
 60. The method of claim 58, wherein at least 50% of the peptide conjugate includes at most one poly(ethylene glycol) moiety.
 61. The method of claim 36, wherein the modified sugar comprises a sugar moiety, and the sugar moiety comprises poly(ethylene glycol).
 62. The method of claim 61, wherein at least 50% of the peptide conjugate includes at least two poly(ethylene glycol) moieties.
 63. The method of claim 61, wherein at least 50% of the peptide conjugate includes at most one poly(ethylene glycol) moiety.
 64. The method of claim 36, wherein the ratio of the sialidase to the peptide is 0.1 U/L:2 mg/mL to 10 U/L:1 mg/mL.
 65. The method of claim 36, further comprising a capping step involving sialylation of the peptide conjugate.
 66. The method of claim 65, wherein a reaction vessel contains the sialidase, the enzyme capable of catalyzing the transfer of a glycosyl linking group, and the modified sugar, and wherein the capping step is performed in the reaction vessel.
 67. The method of claim 66, wherein the enzyme capable of catalyzing the transfer of a glycosyl linking group is a sialyltransferase. 